Anti p irs 1 2y612

Sections of formalin-fixed paraffin embedded (FFPE) xenograft tumors were mounted on slides and examined for proliferating and apoptotic cells by Ki-67 staining and TUNEL assay, respectively, as described previously [47 (link),49 (link)].
IHC staining was performed on paraffin sections using the Novolink^™ Min Polymer Detection System (Leica) as described previously [48 (link),49 (link)]. Briefly, slides were subjected to antigen retrieval using Novocastra Epitope Retrieval Solutions, pH6, followed by incubation with an anti-IRS-1 antibody (1:700, Abcam, ab52167) or anti- p-IRS-1/2Y612 (1:400, Santa Cruz, sc-17195-R) overnight at 4°C. Slides were developed with DAB after incubation with Novolink polymer. Finally, slides were counterstained with hematoxylin. For each sample, ten randomly chosen fields were captured at 20 × magnification and quantified with Imagescope Software (Aperio Technologies, Inc.).

The human colon cancer cell lines, FET, FET-DNRII, CBS, and CBS-RII, were described previously [32 (link)]. All cells were cultured in McCoy’s 5A serum-free medium (Sigma) supplemented with 10 ng/ml epidermal growth factor (EGF), 20 μg/ml insulin, and 4 μg/ml transferrin [44 (link)]. When cells were subjected to growth factor deprivation stress (GFDS), they were cultured in McCoy’s 5A serum-free medium in the absence of EGF, insulin and transferrin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂. TGFβ and the TGFβ RI kinase inhibitor were obtained from R&D Systems and Calbiochem respectively. The following antibodies were used in this study: anti-IRS-1 (1:1000), Cell Signaling #2382, anti-p-IRS-1Y632 (1:600), Santa Cruz sc-17196, anti-p-IRS-1/2Y612 (1:600), Santa Cruz sc-17195-R, anti-Smad3 (1:1000), Cell Signaling #9523, anti-p-Smad3S423/425 (1:1000), Cell Signaling #9520, anti-p-Smad2S465/467 (1:1000), Cell Signaling #3101, anti-XIAP (1:1000), Cell Signaling #14334, anti-Cyclin D1 (1:1000), Cell Signaling #2978 and anti-Actin (1:1000), Sigma #A2066.