Specific Immunoprecipitation Assay Research Articles

Overexpression of ErbB2 in the exocrine pancreas induces an inflammatory response but not increased proliferation

Tyrosine kinase receptors such as members of the epidermal growth factor receptor family and their respective ligands are frequently overexpressed in pancreatic cancer as well as in chronic pancreatitis. In this study, the role of ErbB2 in the exocrine pancreas was examined by ectopic overexpression under the control of the proximal rat elastase promoter. Three independent transgenic mouse lines overexpressing ErbB2 were established by pronuclear injection. Pancreatic mRNA and protein levels were analyzed by real time PCR, immunohistochemistry and immunoblot analysis, RAS activity by using a specific immunoprecipitation assay and various kinase activities by phosphospecific antibodies. Overexpression of ErbB2 in the exocrine pancreas resulted in increased RAS activity and downstream activation of ERK1/2, but not in transgenic increased proliferation of acinar and ductal cells. At later timepoints, some mice showed focal areas of acinar cell damage with upregulated mRNA levels for Cyclin D1 and p16(INK4a). Despite the increased mRNA level, cyclin D1 protein levels were downregulated. We observed areas of focal infiltrations with inflammatory cells interspersed in the exocrine pancreas. NF-kappaB activity was induced in transgenic acinar cells compared with controls contributing to high levels of chemokine and cytokine gene expression such as CCR-1 and CCL3. These data suggest that overexpression of ErbB2 in acinar cells leads to increased RAS activity without cell cycle progression and mediates inflammation via NF-kappaB. We conclude that the biological response of ErbB2/RAS signaling depends highly on cellular context.

The Synthetic Peptide RPRAATF Allows Specific Assay of Akt Activity in Cell Lysates

The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays.

Association of the 72/74-kDa proteins, members of the heterogeneous nuclear ribonucleoprotein M group, with the pre-mRNA at early stages of spliceosome assembly.

We have investigated the role played in precursor mRNA (pre-mRNA) splicing by the protein pair of molecular size 72/74 kDa, which are integral components of a discrete subset of heterogeneous nuclear (hn) ribonucleoproteins (RNPs) named large heterogeneous nuclear RNP (LH-nRNP). This 72/74 kDa pair of proteins has been shown to belong to the hnRNP M group, and are referred to as 72/74(M). By applying specific immunoprecipitation assays in a consecutive series of splicing reactions in vitro, the antigenic 72/74(M) protein species were found to associate with the pre-mRNA and not the intermediate or final products of splicing. Kinetic studies, combined with isolation of pre-spliceosomal and spliceosomal complexes from the splicing reaction, indicated a loose association of 72/74(M) with both the initially formed H assembly and the first splicing-committed E complex. Stable binding was seen at a later stage of the reaction, well in advance of the appearance of the first intermediate products of RNA splicing. Evidence is provided that supports the almost exclusive association of 72/74(M) with pre-mRNA within the pre-spliceosomal A complex. This dynamic binding appeared to involve pre-mRNA sites similar to those of spliceosomal U1 and U2 small nuclear RNP complexes. Moreover, a preferential binding to a truncated RNA containing the 5' exon-intron part, rather than the intron-3' exon part, of pre-mRNA was observed.

Association of the 72/74-kDa proteins, members of the heterogeneous nuclear ribonucleoprotein M group, with the pre-mRNA at early stages of spliceosome assembly

We have investigated the role played in precursor mRNA (pre-mRNA) splicing by the protein pair of molecular size 72/74kDa, which are integral components of a discrete subset of heterogeneous nuclear (hn) ribonucleoproteins (RNPs) named large heterogeneous nuclear RNP (LH-nRNP). This 72/74kDa pair of proteins has been shown to belong to the hnRNP M group, and are referred to as 72/74(M). By applying specific immunoprecipitation assays in a consecutive series of splicing reactions in vitro, the antigenic 72/74(M) protein species were found to associate with the pre-mRNA and not the intermediate or final products of splicing. Kinetic studies, combined with isolation of pre-spliceosomal and spliceosomal complexes from the splicing reaction, indicated a loose association of 72/74(M) with both the initially formed H assembly and the first splicing-committed E complex. Stable binding was seen at a later stage of the reaction, well in advance of the appearance of the first intermediate products of RNA splicing. Evidence is provided that supports the almost exclusive association of 72/74(M) with pre-mRNA within the pre-spliceosomal A complex. This dynamic binding appeared to involve pre-mRNA sites similar to those of spliceosomal U1 and U2 small nuclear RNP complexes. Moreover, a preferential binding to a truncated RNA containing the 5′ exon—intron part, rather than the intron—3′ exon part, of pre-mRNA was observed.

A Specific Immunoprecipitation Assay for the Protein Kinase F A/Glycogen Synthase Kinase 3

A specific immunoprecipitation assay has been developed for the accurate determination of the kinase F A/GSK 3 activity in crude cell preparations. The assay is based on the production and isolation of polyclonal peptide antibodies toward the C-terminus of the rat GSK 3-α and GSK 3-β isoforms. The respective forms of the kinases are captured as active immunocomplexes with immobilized secondary antibodies and their kinase activity toward the synthetic peptide P-GS1 can be accurately measured. Immunoprecipitation with a mixture of the two peptide antibodies allows for the detection and estimation of the total kinase F A/GSK 3 activity in cellular extracts, whereas the α-peptide antibody specifically detects and measures the GSK 3-α isoform. The contribution of GSK 3-β in the total kinase F A/GSK 3 activity of cell fractions can be calculated.

Alternatively spliced variants of the insulin receptor protein. Expression in normal and diabetic human tissues.

Two insulin receptor mRNA transcripts resulting from alternative splicing of exon 11 in the receptor gene are expressed in a highly regulated tissue-specific fashion. To date, there is no information about the relative abundance of the protein isoforms encoded by these mRNAs in tissues of normal or diabetic subjects. We employed an antibody raised against the peptide sequence encoded by exon 11 to develop a specific immunoprecipitation assay that is capable of determining the fraction of receptors that include this amino acid sequence. The assay is based on the relative ability of the exon 11 specific monoclonal antibody (alpha IR alpha) compared to a nonspecific anti-receptor antiserum (B-2) to immunoprecipitate solubilized receptors that are first labeled with 125I-insulin. The assay was validated using standard curves generated with samples composed of known ratios of the two receptor isoforms. Our results in general confirm observations regarding the relative abundance of the two mRNA species in human tissues, with marked predominance of the exon 11+ isoform in liver, and the exon 11- isoform in leukocytes. Similar amounts of both variants are present in placenta, skeletal muscle, and adipose tissue. In studies with this assay using skeletal muscle extracts from control and noninsulin-dependent diabetes mellitus (NIDDM) subjects, as well as in studies of the two mRNAs in control versus NIDDM muscle using a quantitative polymerase chain reaction assay, we could find no significant difference between control and diabetic subjects. This data contradicts a recent report claiming that normal individuals have only the exon 11- mRNA transcript in their skeletal muscle, whereas NIDDM subjects have similar expression of both mRNAs. Given the emerging evidence that functional differences exist between the two receptor isoforms, these studies are relevant to our understanding of insulin receptor function in health and disease.

Heat shock induces two distinct S6 protein kinase activities in quiescent mammalian fibroblasts

The regulation of S6 kinase activity was used to monitor perturbations of intracellular signaling activity during heat shock of quiescent murine and human fibroblasts. Previous reports on exponentially growing insect and plant cells had indicated that 40S ribosomal protein S6 is dephosphorylated during heat shock; thus inhibition of S6 kinase activity by heat shock was anticipated in NIH 3T3 fibroblasts and human cells (HeLa, diploid embryonic fibroblasts MRC-5, and skin-derived fibroblasts). Unexpectedly, two distinct S6 protein kinases were activated in quiescent fibroblasts after heat exposure. One of the enzymes was partially purified by sequential column chromatography and was determined to be equivalent to the enzyme activated by serum and other growth factors, referred to here as pp70-S6 protein kinase. The other protein S6 kinase, pp90rsk, was identified by a specific immunoprecipitation assay. Monitoring both enzymatic activities during heat shock revealed a temporal pattern of activation that was reversed when compared to non-stressed, mitogen-stimulated cells. Finally, heat shock stimulated protein S6 phosphorylation in cultured, quiescent mammalian cells. These data demonstrate that specific protein kinases can be activated during heat shock, and that some early mitogenic signals may also participate in the response of cells to physiologic stress.

Measurement of human nerve growth factor binding sites in brain and in peripheral tissues by a specific immunoprecipitation assay

Using a monoclonal antibody against the human nerve growth factor (NGF) receptor (20.4 IgG), a specific immunoprecipitation assay for the quantitation of human NGF binding sites has been established. The procedure involves specific labeling of NGF receptors by covalent crosslinking to 125I-NGF with a carbodiimide reagent, and subsequent immunoprecipitation of the detergent-extracted 125I-NGF-receptor complexes with the 20.4 IgG. This two-site assay provides a specific method to measure NGF binding sites in peripheral tissues and central nervous system. Moreover, it allows analysis of the immunoprecipitated receptor species by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The finding that specific NGF binding sites are expressed on human neuronal and nonneuronal tissues, including lymphoid tissues, indicates that NGF exerts a broader physiological function than originally ascribed.

Monoclonal antibodies against an intracellular phospholipase A2 from rat liver and their cross-reactivity with other phospholipases A2.

The membrane-associated phospholipase A2 from rat liver mitochondria was solubilized and partially purified by AcA 54 gel filtration and Matrex gel blue A chromatography. The approximately 2500-fold purified preparation was injected into mice to prepare monoclonal antibodies against phospholipase A2 after fusion of spleen cells and mouse SP2/0 myeloma cells. Hybridoma supernatants were assayed for antibody production in enzyme-linked immunosorbent assay with partially purified phospholipase A2 as antigen. Positive clones were tested for their ability to bind phospholipase A2 in a specific immunoprecipitation assay involving protein-A--Sepharose to which rabbit anti-(mouse immunoglobulins) and monoclonal antibodies from hybridoma supernatants were complexed. Twelve clones producing antibodies that bound mitochondrial phospholipase A2 were identified. The binding of all of these antibodies to protein fractions eluted from AcA 54 and Matrex gel blue A columns coincided with the phospholipase A2 activity in these fractions. All monoclonal antibodies showed cross-reactivity with rat liver cytosolic and solubilized rat platelet phospholipase A2. Extracellular phospholipase A2 from rat and pig pancreas or Crotalus atrox were not recognized by the anti-(mitochondrial phospholipase A2) antibodies.

Expression of Vitiligo Antigen on a Revertant Line of Hamster Melanoma Cells

Our laboratory has recently reported that over 80% of patients with common vitiligo have circulating antibodies to cell-surface antigens on normal human melanocytes. The slow growth rate of these cells limits the assays that can be performed for antibody detection. We now have found that the antigens defined by vitiligo sera on melanocytes are also expressed on FF cells, a revertant line of hamster melanoma cells. These antigens can be detected both by indirect immunofluorescence and specific immunoprecipitation assays. The presence of "vitiligo" antigens on hamster FF cells will aid further study of the abnormal immune response in vitiligo.

Yeast super-suppressors are altered tRNAs capable of translating a nonsense codon in vitro.

tRNA isolated from two different yeast super-suppressor strains translates a known nonsense mutation in vitro, whereas tRNA from a closely related nonsuppressing strain does not. Suppression was assayed by translation of RNA isolated from an amber coat mutant of bacteriophage Qbeta (GB11) in a protein-synthesizing system derived from mouse tissue culture cells (L cells). Suppressed forms of Qbeta coat protein synthesized in vitro were quantitatively detected by a specific immunoprecipitation assay. The L-cell protein-synthesizing system also responds to E. coli suppressor tRNA. This indicates that the biochemical mechanism for nonsense suppression is very similar in yeast and E. coli. These findings also provide additional evidence that the amber codon (UAG) functions as one of the mammalian chain-terminating codons. Since the suppression assay utilizes protein-synthesizing components isolated from mammalian cells, it should prove useful in the search for mammalian nonsense suppressors.