Rabbit anti fak

Equal amount of proteins from each sample were loaded and run on SDS-PAGE gels (BioRad, Philadelphia, PA, USA) and then transferred to the methanol-activated Hybond P 0.45 PVDF membranes (GE Healthcare Biosciences, Pittsburgh, PA, USA). Membrane blocking was performed with a TBS-T buffer containing 5% skimmed milk and followed by incubation with rabbit anti-FAK (Cell Signalling Technology, Danvers, MA, USA), rabbit anti-P21 (Cell Signalling Technology), rabbit anti-P16 (Cell Signalling Technology), rabbit anti-pFAK (Y397 Thermo Fisher, Waltham, MA, USA), mouse anti-Akt (BD Bioscience, San Jose, CA, USA), mouse anti-pAkt (S473 BD Bioscience, San Jose, CA, USA), rabbit anti-β-Actin (Cell Signalling Technology), and rabbit anti-α-Tubulin (Cell Signalling Technology) antibodies at 1:1000 dilution for 2 h at RT. After washing, antigen–antibody reactions were developed using anti-mouse or anti-rabbit Horseradish Peroxidase (HRP)-conjugated secondary antibodies (DAKO, Glostrup, Denmark) for 60 min at RT. Finally, the protein bands were visualised using an electrogenerated chemiluminescence (ECL) detection system. The amount of target protein was normalised to the structural protein (β-Actin or α-Tubulin). Adobe Photoshop CC version 2017.0.020161012.r.53X64 was used to compare the intensity of protein bands.

Cells were seeded on 12-mm diameter glass coverslips and when they had reached approximately 60–70% confluence, cells were fixed for 30 min with 4% paraformaldehyde, blocked with TBS-containing donkey serum (10%) and incubated overnight at 4 °C with mouse anti-CB (1:1000, Swant) and rabbit anti-FAK (1:50; Cell Signaling Technology, Danvers, MA, USA) antibodies diluted in Tris-buffered saline (TBS 1X). After washing, cells were incubated for 3 h at room temperature with the following secondary antibodies: Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:100; Jackson Immunoresearch Laboratories, West Grove, PA, USA) and Cy3-conjugated donkey anti-mouse (IgG) (1:100; Jackson Immunoresearch Laboratories). 4′,6-diamidino-2-phenylindole (DAPI; 5 μg/mL; Molecular Probes, Eugene, OR, USA) was used to stain nuclear DNA and coverslips were mounted with Hydromount solution (National Diagnostics, Atlanta, GA, USA). Images were acquired using a LEICA fluorescent microscope DM6000B (Wetzlar, Germany) equipped with a Hamamatsu camera C4742-95 (Bridgewater, NJ, USA).

Whole cells were lysed with RIPA lysis buffer (Beyotime, China, Cat No.P0013B). After protein quantification with the BCA Protein Assay Kit (Thermo Fisher Scientific, CA, Cat No.23225), equal amounts of proteins were separated on SDS-PAGE, and transferred to a PVDF membrane. Antibodies against the following proteins were used: Rabbit-anti-NRF2, rabbit-anti-MLC2, rabbit-anti-phospho-MLC2 (Thr18/Ser19), rabbit-anti-FAK, rabbit-anti-phospho-FAK (Tyr397), and rabbit-anti-ERR1, all purchased from Cell Signaling (Cat No. 12721, 3672, 3674, 3285, 8556 and 13826, respectively). Mouse-anti-RhoA was purchased from Cytoskeleton (Cat No. ARH03, CA), and mouse-anti-actin was purchased from ProteinTech (Cat No. 60008-1-Ig, CA).

Cell extracts were prepared by adding lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 0.5% Nonidet-40, 1 mM dithiothreitol) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Cells were removed with a cell scraper, incubated with lysis buffer at 4 °C for 2 h and then centrifuged at 16100 × g. Supernatants were collected and treated with SDS sample buffer. Western blot analyses were performed with rabbit anti-FAK (1:1000, Cell Signaling) and rabbit anti-tubulin (1:2000, Cell Signaling) antibodies. The peroxidase-conjugated anti-rabbit IgG was used as secondary antibody. ImageJ software (https://imagej.nih.gov/ij/) was used for the quantitative analysis and different gel images were quantified using linear signal ranges.

Cells were seeded at 4 × 10⁵ per well in six-well plates with appropriate growth medium and incubated at 37 °C, 5% CO₂ overnight, or treated with drugs for indicated period of time before harvesting. Cell lysates were prepared by cell lysis on ice with 1X RIPA buffer (Cell Signaling, #9806) containing protease inhibitor (Roche, #11836170001) and phosphatase inhibitor cocktail (Sigma, #P5726). Immunoblotting of individual protein bands was performed by incubating the PVDF membranes with the following primary antibodies (all purchased from Cell Signaling) diluted 1:1000 in 5% BSA/TBST: rabbit anti-phospho-ERK (#9101), mouse anti-ERK1/2 (#9107), rabbit anti-phospho-AKT (Ser473) (#4060), mouse anti-AKT (#2920), rabbit anti-phospho-FAK (Y397) (#8556), rabbit anti-FAK (#13009), and rabbit anti-GAPDH (#2118). Fluorescent Alexa 488-conjugated donkey anti-rabbit or anti-mouse antibodies (Life Technologies, A21206 or A21202) were used as secondary antibodies diluted 1:5000 in 5% BSA/TBST to visualize the protein bands on the Pharos Molecular Imager (BioRad). Intensities of individual protein bands from the scanned images were measured using ImageJ after subtracting background. Phosphoprotein levels were then normalized to either GAPDH or total level of the corresponding protein. Uncropped images of immunoblots are shown in Supplementary Figures 7 to 11.