Immunoprecipitation Procedure Research Articles

Fluorescence ImmunoPrecipitation (FLIP): a Novel Assay for High-Throughput IP.

BackgroundThe immunoprecipitation (IP) assay is a valuable molecular biology tool applied across a breadth of fields. The standard assay couples IP to immunoblotting (IP/IB), a procedure severely limited as it is not easily scaled for high-throughput analysis.ResultsHere we describe and characterize a new methodology for fast and reliable evaluation of an immunoprecipitation reaction. FLIP (FLuorescence IP) relies on the expression of the target protein as a chromophore-tagged protein and couples IP with the measurement of fluorescent signal coating agarose beads. We show here that FLIP displays similar sensitivity to the standard IP/IB procedure but is amenable to high-throughput analysis. We applied FLIP to the screening of mouse monoclonal antibodies of unknown behavior in IP procedures. The parallel analysis of the considered antibodies using FLIP and IP/western shows good correlation between the two procedures. We also show application of FLIP using unpurified antibodies (hybridoma supernatant) and we developed a publicly available tool for the easy analysis and quantification of FLIP signals.ConclusionsAltogether, our characterizations of this new methodology show that FLIP is an appealing and reliable tool for any application of high-throughput IP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-016-0046-x) contains supplementary material, which is available to authorized users.

Immunoprecipitation of Cullin-RING Ligases (CRLs) in Arabidopsis thaliana Seedlings.

CRL (Cullin-RING ubiquitin ligase) is the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-RING Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure.

In vitro Reconstitution Assay of miRNA Biogenesis by Arabidopsis DCL1.

microRNAs (miRNAs) are small non-coding RNAs, regulating most if not all, biological processes in eukaryotic organisms. miRNAs are initially processed from primary transcripts (pri-miRNAs) to produce miRNA precursors (pre-miRNAs), that are further processed into miRNA and its complementary strands (miRNA/*). In Arabidopsis, and possibly other plants, the processing from pri-miRNAs to pre-miRNAs and from pre-miRNAs to miRNA/* are both implemented through Dicer-like 1 (DCL1) complexes. Recently, we demonstrated isolation of DCL1 complexes of unprecedented quality from in planta. We further successfully reconstituted DCL1 cleavage assays in vitro that were able to fully recapitulate in vivo miRNA biogenesis. Here we provide a detailed protocol of DCL1 reconstitution assays. The protocol comprises three major parts (Figure 1): 1) Preparation of pri- and pre-miRNA transcripts (Procedures A-C); 2) Purification of the recombinant Arabidopsis DCL1 machinery from Nicotiana benthamiana (N. benthamiana) through immunoprecipitation (IP) (Procedures D and E); and 3) in vitro processing of radioisotope-labeled pri- or pre-miRNAs using the isolated DCL1 complexes (Procedure F). It is our desire that the protocol be a powerful tool for the RNAi community to study mechanistic issues or to develop RNA silencing technologies.

Enrichment of low-abundant protein targets by immunoprecipitation upstream of mass spectrometry.

Immunoprecipitation (IP) is commonly used upstream of mass spectrometry (MS) as an enrichment tool for low-abundant protein targets. However, several aspects of the classical IP procedure such as nonspecific protein binding to the isolation matrix, detergents or high salt concentrations in wash and elution buffers, and antibody chain contamination in elution fractions render it incompatible with downstream mass spectrometry analysis. Here, we discuss two IP workflows that are designed to minimize or eliminate these contaminants: the first employs biotinylated antibodies and streptavidin magnetic beads while the second method utilizes a traditional antibody that is oriented and cross-linked to Protein AG magnetic beads. Both modified magnetic supports have low background binding and both antibody immobilization strategies significantly reduce or eliminate antibody heavy and light chain contamination in the eluent, minimizing potential ion suppression effects and thereby maximizing detection of target antigens and interacting proteins.

Immediate chromatin immunoprecipitation and on-bead quantitative PCR analysis: a versatile and rapid ChIP procedure.

Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana.

A robust chromatin immunoprecipitation protocol for studying transcription factor-DNA interactions and histone modifications in wood-forming tissue.

Woody cells and tissues are recalcitrant to standard chromatin immunoprecipitation (ChIP) procedures. However, we recently successfully implemented ChIP in wood-forming tissue of the model woody plant Populus trichocarpa. Here we provide the detailed ChIP protocol optimized for wood-forming tissue that we used in those studies. By using stem-differentiating xylem (SDX; a wood-forming tissue), we identified all steps that were ineffective in standard ChIP protocols and systematically modified them to develop and optimize a robust ChIP protocol. The protocol includes tissue collection, cross-linking, nuclear isolation, chromatin extraction, DNA fragmentation, immunoprecipitation, DNA purification and sequence analysis. The protocol takes 2.5 d to complete and allows a robust 8-10-fold enrichment of transcription factor (TF)-bound genomic fragments (~150 ng/g of SDX) over nonspecific DNAs. The enriched DNAs are of high quality and can be used for subsequent PCR and DNA-seq analyses. We used this protocol to identify genome-wide specific TF-DNA interactions during wood formation and histone modifications associated with regulation of wood formation. Our protocol, which may be suitable for many tissue types, is so far the only working ChIP system for wood-forming tissue.

Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody.

Human parainfluenza virus 3 (HPIV3) commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs) have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of MAbs against hemagglutinin-neuraminidase (HN) of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting, and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. The newly-developed MAbs could thus be a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.

Retrotransposon Alu is enriched in the epichromatin of HL-60 cells

Epichromatin, the surface of chromatin facing the nuclear envelope in an interphase nucleus, reveals a “rim” staining pattern with specific mouse monoclonal antibodies against histone H2A/H2B/DNA and phosphatidylserine epitopes. Employing a modified ChIP-Seq procedure on undifferentiated and differentiated human leukemic (HL-60/S4) cells, >95% of assembled epichromatin regions overlapped with Alu retrotransposons. They also exhibited enrichment of the AluS subfamily and of Alu oligomers. Furthermore, mapping epichromatin regions to the human chromosomes revealed highly similar localization patterns in the various cell states and with the different antibodies. Comparisons with available epigenetic databases suggested that epichromatin is neither “classical” heterochromatin nor highly expressing genes, implying another function at the surface of interphase chromatin. A modified chromatin immunoprecipitation procedure (xxChIP) was developed because the studied antibodies react generally with mononucleosomes and lysed chromatin. A second fixation is necessary to securely attach the antibodies to the epichromatin epitopes of the intact nucleus.

Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

Global analysis of transcription factor-binding sites in yeast using ChIP-Seq.

Transcription factors influence gene expression through their ability to bind DNA at specific regulatory elements. Specific DNA-protein interactions can be isolated through the chromatin immunoprecipitation (ChIP) procedure, in which DNA fragments bound by the protein of interest are recovered. ChIP is followed by high-throughput DNA sequencing (Seq) to determine the genomic provenance of ChIP DNA fragments and their relative abundance in the sample. This chapter describes a ChIP-Seq strategy adapted for budding yeast to enable the genome-wide characterization of binding sites of transcription factors (TFs) and other DNA-binding proteins in an efficient and cost-effective way.Yeast strains with epitope-tagged TFs are most commonly used for ChIP-Seq, along with their matching untagged control strains. The initial step of ChIP involves the cross-linking of DNA and proteins. Next, yeast cells are lysed and sonicated to shear chromatin into smaller fragments. An antibody against an epitope-tagged TF is used to pull down chromatin complexes containing DNA and the TF of interest. DNA is then purified and proteins degraded. Specific barcoded adapters for multiplex DNA sequencing are ligated to ChIP DNA. Short DNA sequence reads (28-36 base pairs) are parsed according to the barcode and aligned against the yeast reference genome, thus generating a nucleotide-resolution map of transcription factor-binding sites and their occupancy.

Rapid purification of Soybean mosaic virus from small quantities of tissue by immunoprecipitation

A rapid, simple, and efficient method for purification of Soybean mosaic virus (SMV) was developed based on immunoprecipitation. Traditional centrifugation-based methods for purification of SMV and other potyviruses require long, complicated procedures and large quantities of infected tissue (100–500g). The immunoprecipitation procedure described in this study allows the purification of intact SMV virion particles in 4h from 0.5g of tissue. The reliability of this procedure was demonstrated by RT-PCR and transmission electron microscopy (TEM). This method will be useful for high-throughput examination of the physical and morphological properties of virus particles because intact virion preparations ready for TEM observation can be purified rapidly from very small tissue samples.

The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components

Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status.

Abstract 2209: Cellular characterization of chromatin state via high throughput ChIP assay

Abstract Expression of eukaryotic genes during development requires complex spatial-temporal regulation. This complex regulation is often achieved through the coordinated interaction of transcription regulatory elements in the promoters of the target genes. The identification and mapping of regulatory elements in genome scale is crucial to understand how gene expression is regulated. Chromatin immunoprecipitation is a standard method for assessing the occupancy of DNA binding proteins in vivo in their native chromatin context using antibodies. However, standard chromatin immunoprecipitation procedure is time consuming, labor intensive and not suited for analyzing many samples simultaneously. Recently, we have developed a simple high throughput chromatin immunoprecipitation protocol that utilizes high capacity protein A/G coated magnetic beads and 96 well plates. This protocol requires fewer steps and less hands-on time, has higher sensitivity and more reliable performance as compared to conventional approaches. The method is also compatible with multi-channel pipetting and liquid handling systems. We have successfully used this protocol to map various clinically relevant chromatin marks and controls across several cell types to quantitatively measure chromatin states. This analysis included a variety of marks corresponding to repressed, poised and active promoters, strong and weak enhancers, putative insulators, transcribed regions, as well as large-scale repressed and inactive domains. This study demonstrates the utility of this approach for the characterization of model cellular systems in perturbation studies with chemical probes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2209. doi:1538-7445.AM2012-2209

Native Accessible ChIP: A Novel Approach to Interrogate Protein‐DNA Interactions in Accessible Chromatin

We have developed a novel technology for isolating accessible chromatin. When cells are permeabilized and treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin can be easily isolated and characterized. We now report that the isolated accessible chromatin is still associated with its cognate DNA‐binding proteins and histones. Based on this, we developed a novel chromatin immunoprecipitation (ChIP) procedure that does not require protein‐DNA cross‐linking or DNA shearing. Using this method, termed NA‐ChIP (for native accessible ChIP), we demonstrate that RNA polymerase II (RNAPII), TBP and H3K4me3 are all associated with active promoters, but are rarely detected at silenced promoters. Comparison of NA‐ChIP and a standard ChIP assay shows that immunoprecipitation (IP) efficiency is similar, but IP specificity is dramatically different. To measure IP specificity we compared the ratio of active promoter DNA precipitated with RNAPII (signal) relative to inactive promoter DNA precipitated (noise). We find that NA‐ChIP is highly specific with a signal‐to‐noise ratio >500; standard ChIP is less specific with a signal‐to‐noise ratio of 9. We conclude that NA‐ChIP offers significant advantages over standard ChIP, including dramatically reduced background and an easier workflow.

Nanodisc-based Co-immunoprecipitation for Mass Spectrometric Identification of Membrane-interacting Proteins

Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.

Abstract 710: Gene expression profile of the Wnt signaling pathway in trastuzumab resistant breast cancer cells

Abstract Introduction: We are interested in understanding mechanisms that lead to Trastuzumab (Herceptin) resistance in HER2 overexpressing breast tumors. In this study, we examined the role of the Wnt/β-catenin signaling pathway in Trastuzumab resistance. In addition to the Wnt pathway genes, we examined gene expression profiles using 44K gene chip array and EMT associated genes in Trastuzumab resistant cells. Methods: Wnt pathway gene profiles were examined by Agilent chip (4×44K) and Wnt/β-catenin specific array. HER2 overexpressing breast cancer cells, SKBR3 and trastuzumab resistant clone, SKBR3 100-8 cells were used as cell models. The EMT (Epithelial to Mesenchymal Transition) markers Snail, Slug, Twist and E-Cadherin were examined by both real-time PCR and western blot analysis. Cell invasiveness was determined by invasive assay. HER2-HER3 dimerization was examined using immunoprecipitation and immunoblotting procedures. Results: We established a Trastuzumab resistant cell line from the HER2 overexpressing parental SKBR3 cell line. The resistant clone called SKBR3/100-8 is insensitive to 100µg/ml of Trastuzumab. It has a mesenchymal or more basal-like morphology. It overexpresses HER2 protein and mRNA more than the parental line SKBR3. This resistant clone shows increased dimerization of HER2 with HER3. The SKBR3 100-8 cells were found to overexpress EGFR1. It is more invasive compared to wt SKBR3 and shows a significant increase in EMT marker, SLUG, but a decrease in Snail and E-cadherin compared to the wtSKBR3 cells. The resistant clone shows increase in phospho-Akt, pGSK3β and a decrease in β-Catenin. Agilent chip (4×44K) array (containing 84 Wnt pathway genes) identified the following Wnt pathway genes to be upregulated by 2 or more fold in the resistant clone: AXIN1, CSNK1A1, CSNK1D, CTBP2, DAAM1, c-JUN, TLE1, WNT3 and WNT6. Conversely, the following genes were downregulated by 2 or more fold in the resistant clone: AES, CCND3, CTNNB1, FZD1, FZD4, FZD5, PITX2, PPP2CA, PPP2R1A, WIF1, WNT11, and WNT4. The resistant clone also increased Nanog and VEGF significantly. TGFβ treatment in wtSKBR3 increased SLUG, Snail, Twist, and decreased E-Cadherin. The cells showed more mesenchymal morphology than wtSKBR without TGFβ treatment. TGFβ treatment in wtSKBR3 upregulated: c-Jun, C-Myc, CCND1, FosL1, LEF1, and TCF-1. TGFβ also induced Met, MMP1, MMP7, MMP9, MMP26, Nanog, NOS2, POU5F1, PPARd, Survivin, uPAR and VEGF. In contrast, TGFβ upregulated only c-Jun, Met, Nanog, and VEGF in SKBR3 100-8 cells. Conclusion: Wnt/β-catenin signaling is highly involved in the trastuzumab resistant breast cancer cells. The resistant signature involves c-Jun, Met, Nanog and VEGF. These results suggest a very specific signature that needs further studies for target therapy for the Trastuzumab resistant tumors and implies the use of anti VEGF therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 710. doi:10.1158/1538-7445.AM2011-710

Abstract 3011: Evaluation of a high throughput platform for chromatin immunoprecipitation

Abstract Expression of eukaryotic genes during development requires complex spatial-temporal regulation. This complex regulation is often achieved through the coordinated interaction of transcription regulatory elements in the promoters of the target genes. The identification and mapping of regulatory elements in genome scale is crucial to understand how gene expression is regulated Chromatin immunoprecipitation is a standard method for assessing the occupancy of DNA binding proteins in vivo in their native chromatin context using antibodies. However, standard chromatin immunoprecipitation procedure is time consuming, labor intensive and not suited for analyzing many samples simultaneously. Recently, we have developed a simple chromatin immunoprecipitation protocol, which utilizes high capacity protein A/G coated magnetic beads and 96 well plate. This protocol requires fewer steps, little hands-on time and is compatible with multi-channel pipetting and liquid handling robot. This protocol allows genome wide mapping of transcription regulatory network in a high throughput manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3011. doi:10.1158/1538-7445.AM2011-3011

Two Distinct Amyloid β-Protein (Aβ) Assembly Pathways Leading to Oligomers and Fibrils Identified by Combined Fluorescence Correlation Spectroscopy, Morphology, and Toxicity Analyses

Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ∼330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ∼128 kDa (∼32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aβ(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial.

Orderly Inactivation of the Key Checkpoint Protein Mitotic Arrest Deficient 2 (MAD2) during Mitotic Progression

Anaphase is promoted by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) only when all the chromosomes have achieved bipolar attachment to the mitotic spindles. Unattached kinetochores or the absence of tension between the paired kinetochores activates a surveillance mechanism termed the spindle-assembly checkpoint. A fundamental principle of the checkpoint is the activation of mitotic arrest deficient 2 (MAD2). MAD2 then forms a diffusible complex called mitotic checkpoint complex (designated as MAD2(MCC)) before it is recruited to APC/C (designated as MAD2(APC/C)). Large gaps in our knowledge remain on how MAD2 is inactivated after the checkpoint is satisfied. In this study, we have investigated the regulation of MAD2-containing complexes during mitotic progression. Using selective immunoprecipitation of checkpoint components and gel filtration chromatography, we found that MAD2(MCC) and MAD2(APC/C) were regulated very differently during mitotic exit. Temporally, MAD2(MCC) was broken down ahead of MAD2(APC/C). The inactivation of the two complexes also displayed different requirements of proteolysis; although APC/C and proteasome activities were dispensable for MAD2(MCC) inactivation, they are required for MAD2(APC/C) inactivation. In fact, the degradation of CDC20 is inextricably linked to the breakdown of MAD2(APC/C). These data extended our understanding of the checkpoint complexes during checkpoint silencing.

Polypyrimidine Tract-Binding Protein 2 Binds to Selective, Intronic Messenger RNA and MicroRNA Targets in the Mouse Testis

Here we use an in vivo cross-linking and immunoprecipitation procedure to detect RNA targets of the multifunctional RNA-binding protein polypyrimidine tract-binding protein (PTBP) 2 in mouse testis. Eleven known mRNAs, including Ptbp2 mRNA, 28 RNAs matching intron sequences, and 12 small RNAs and repeat sequences are identified. The specificity of interaction between PTBP2 and its target RNAs was confirmed using RNA interference with mouse N2A cells. Reduction of PTBP2 levels led to decreases in 7 of 10 of the mRNAs, to the repression of alternative splicing of introns, and to reductions in specific miRNAs.