Supplemental Figure Legends from Activator Protein-1 Has an Essential Role in Pancreatic Cancer Cells and Is Regulated by a Novel Akt-Mediated Mechanism

Abstract

<p>Supplementary Figure. 1A. Nuclear extracts (N.E.) from various serum-starved pancreatic cancer cell lines were probed for NF-κBbinding activity. Also included were serum-starved Panc-28 cells after stimulation by IL-1. Supershift assays were performed with unstimulated Panc-28 cell extracts using anti-p65/NFκB and nonspecific (IgG) antibodies. Supplementary Figure. 1B Whole-cell extracts were prepared from untreated or TNF-treated cells and then incubated with anti- JNK1/2 antibodies. Immune complexes were washed several times with a lysis buffer and assayed for JNK kinase activity with 2 μg of GST-c-Jun (amino acids 1-79) used as substrate. Assay mixtures, which included 0.2 mM [γ-32P] ATP and 10 mM MgCl2, were incubated for 5 min at 30 {degree sign}C, at which point the reactions were terminated by the addition of a sodium dodecyl sulfate sample buffer. Radiolabeled GST- c-Jun bands were detected by autoradiography. Serum-starved Panc-28 cells were treated with or without the JNK inhibitor SP600125 for 2 h. Whole-cell extracts were probed by Western blotting for c-Jun and phospho-c-Jun (Ser63). Supplementary Figure. 1C. A collagenase/luciferase reporter gene with (-73 Col/luc) or without (-60 Col/luc) an AP-1-binding site was cotransfected with the indicated constructs into c-Jun-/- MEFs stably expressing the pMX vector, c- Jun-TA or c-Jun SAA/TA. Sixteen to twenty four hours after transfection, cells were serum-starved overnight and either harvested right away or stimulated with PDGF-BB (10 ng/ml). After PDGF treatment (24 h) cells were lysed and protein extracts assayed for luciferase activity. Each data point was obtained in triplicate and used to calculate the averages and standard deviations.</p>