Itgav

Western blotting was performed with the following primary antibodies; Erk (Cell Signalling 4695, Beverly, MA, http://www.cellsignal.com), Jun (Thermo‐Scientific PA1‐833), Itgav (BD Bioscience, San Diego, http://www.bdbiosciences.com; 550024), Itgb7 (Santa Cruz, Santa Cruz, CA, http://www.scbt.com; 15330), and fibronectin (Sigma F3648). Secondary antibodies were IRDye 680LT anti‐Rabbit IgG and IRDye 680LT anti‐Rat IgG (Li‐Cor Biosciences). Signal was detected and quantified using the Odyssey Imaging System (Licor Biosciences). All Western blots are representative of at least three biological replicates.

For immunoblotting 2.5 × 10⁵ to 1 × 10⁶ cells were lysed in laemmli buffer containing 5% 2-β-mercaptoethanol (Sigma Aldrich, Germany), sonicated and boiled for 5 min at 95 °C. Proteins were size fractionated on prestained gradient polyacrylamide gels (Mini-PROTEAN^®TGX Stain-Free™ Precast Gels, Biorad, Germany), blotted onto 0.2 μm PVDF membrane, blocked 2 h in 5% low fat milk powder and incubated overnight with primary antibodies against ITGAV (BD Biosciences, Germany), ITGA5 (Biolegend, USA), AKT, phAKT (S473), phERK (Thr202/Tyr204), ERK, PLIN1, FABP4, YAP, TAZ (all from Cell signaling, USA), p21^Cip1 (ThermoScientific, Austria), survivin (Enzo Lifesciences, Switzerland), p73 (Imgenex, USA), p53 (MyBiosource, USA), GAPDH (6c5,Santa Cruz, Germany). After washing, horseradish peroxidase-conjugated sheep-anti-mouse and sheep-anti-rabbit antibodies (all from Cell signaling, USA) were incubated for 1 h and the reaction was visualized by enhanced chemiluminescence reagent ECL (Biorad, Germany) using a Biorad ChemidocMP gel analyzer for detection. Quantification was performed with the ImageLab 5.0 software (Biorad, Germany) according to the manufacturer’s instructions. Total protein loading was visualized using the ChemidocMP gel analyzer and employed as an internal loading control for all immunoblots59 (link).