Met d1c2

Three μm paraffin-embedded tissue sections were deparaffinized and rehydrated, followed by blocking the endogenous peroxidase with 0.3% hydrogen peroxidase. After Heat Induced Antigen Retrieval (HIER) for 10 minutes in 10 mmol/L citrate buffer (pH 6.0), the tissue sections were incubated with primary rabbit mAb Met (D1C2), Cell Signaling, MA, USA, dilution 1:300, incubation time 1 h) and Ki67 mouse mAb (Dako, Glostrup, Denmark, dilution 1:100, incubation time 30 min) followed by treatment with Ultravision Labeled Horseradish peroxidase (HRP) polymer (UVLP, Dako, Glostrup, Denmark, incubation time 15 min). Antibody binding was visualized with DAB+ chromogen and counterstained with Heamatoxylin.

Phospho-MET (Tyr1234/35) (#3126), total MET (#4560), and Pathscan multiplex western cocktail I (phospho-p90RSK, phospho-AKT, phospho-p42/44 MAPK, phospho-S6, Rab11) (#5301) Met (D1C2) (#8198), Caspase-3 (#9662) AKT (#9272), eIF2 α (#9722), phospho-eIF2α (Ser51) (#9721), antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin (#MAB1501) antibody was from (Millipore Corporation, USA) and ERK 1 (K-23) (#sc-94) from (Santa Cruz Biotechnology, Santa Cruz, USA).

UWB1.289 and UWB1.289+BRCA1 cells were collected by scraping on ice, and Western blot analyses were performed using TGX stain-free gels (Bio-Rad Laboratories). Following blocking in 5% milk on a shaker at 4 °C overnight, membranes were incubated with primary antibodies against SOX2 (D6D9, Cell Signaling), Nanog (CL5810, Atlas Antibodies), OCT4 (NB100-2379, Novus Biologicals, Abingdon, UK), RAD51 (14B4, Abcam, Cambridge, UK), BRCA1 (#9010, Cell Signaling, Danvers, MA, USA), and Met (D1C2, Cell Signaling) on a shaker overnight at 4 °C. The primary antibody HSP90 (68/HSP90, BD Biosciences, San Jose, CA, USA), used to ensure equal protein loading on gels, was applied to the membranes for 2 h at room temperature. The membranes were washed and incubated with HRP-conjugated secondary anti-rabbit or anti-mouse antibodies (Thermo Fisher Scientific) for 1 h at room temperature. Detection was performed with Luminata Forte Western HRD Substrate (Merck-Millipore, Burlington, MA, USA) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, CA, USA), using Bio-Rad’s Stain-Free imaging technology (Bio-Rad, Hercules, CA, USA). Quantification of Western blot results was performed using ImageLab^® (Bio-Rad), and intensity was normalized to stain-free blots.