P egfr

For Western blot analysis, cells were lysed with RIP buffer (Boster) supplemented with RNase inhibitor and phosphatase inhibitor after cell transfection. Protein concentration was identified using a BCA kit (Invitrogen). The lysate sample was separated on a 10% SDS-PAGE gel and blotted onto blotted onto PVDF membranes. The membrane was incubated with primary antibody overnight at 4 °C, including EGFR (1:2000, Abcam), P-EGFR (1:2000, Abcam), KRAS (1:2000, Abcam), BRAF (1:2000, Abcam), MEK1/2 (1:2000, Abcam), P-MEK1/2 (1:1000, Proteintech), ERK (1:2000, Abcam), P-ERK (1:2000, Abcam), MAP3K1 (1:700, Proteintech), P-MAP3K1 (1:1000, Proteintech), β-actin (1:700, Proteintech), incubate for 2 hours at room temperature with anti-rabbit secondary antibody. Finally, protein band detection was performed using a Chemiluminescent Reagent (ECL) kit (Beyotime Biotechnology).

Cardiomyocytes and cardiac fibroblasts were collected and lysed to extract the proteins. The membrane proteins were extracted with a cell membrane protein extraction kit (Biyuntian Institute of Biotechnology, Shanghai, China) following the manufacturer's instructions. The protein concentration was detected using a BCA assay. The proteins were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Then, the PVDF membranes were blocked with 5% skim milk for 1 h and incubated with antibodies against MR (1 : 60, Abcam), EGFR (1 : 1200, Abcam), pEGFR (1 : 800, Abcam), ERK1/2 (1 : 1000, Abcam), pERK1/2 (1 : 600, Cell Signaling Technology, Inc., USA), Na-K ATPase (1 : 1000, Abcam), and β-actin (1 : 1000, Abcam) overnight at 4°C. After the membranes were washed with TBST, they were incubated with secondary antibodies for 4 h at room temperature. Finally, immunoreactivity was visualized with an ECL kit (Millipore, MA, USA). The relative expression levels (ratio compared to Na-K ATPase or β-actin expression) of the target proteins were quantified by ImageJ software.

Cells on SISmuc or in Petri dishes were washed twice with ice-cold PBS and lysed (30 min, 4 °C) on a rocking platform with modified RIPA buffer containing 137 mM NaCl, 50 mM NaF, 20 mM Tris (pH 8), 2 mM EDTA, 10% (v/v) glycerol, 1.0% (v/v) NP40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, and freshly added 1 mM Na3VO4 and protease inhibitor cocktail tablets (cOmplete™ mini EDTA-free, Roche, Basel, Switzerland). The lysate was centrifuged (14,000 rpm, 10 min, 4 °C) and the supernatant transferred into clean tubes for quantitation of the protein concentration using DC Protein Assay (500-0116, BioRad, Hercules, CA, USA) or stored (−80 °C) until further use. Before SDS gel electrophoresis, a protein precipitation with methanol/chloroform according to Wessel and Flügge [68 (link)], was performed. Antibodies used for WB: EGFR (4267, CST), p-EGFR (Tyr1068, Y68) (32430, Abcam), Met (8198, CST), p-Met (Tyr1234/1235) (3077, CST, Danvers, MA, USA), Erk1/2 (05-1152, Millipore, Burlington, MA, USA), p-Erk1/2 (Thr202/Tyr204) (4370, CST), Akt (pan) (4691, CST), p-Akt (Ser473) (4060, CST), α-Tubulin (3873, CST). We used the Protein Ladder_ProSieve QuadColor Protein Marker (Lonza, Basel, Switzerland).

A431 ctr or KO cells were lysed on ice in NP-40 lysis buffer (100 mM NaCl 100 mM Tris pH8, 1% NP-40) for 15 min. Pellets were discarded after spinning at max speed for 15 min at 4 °C and supernatant was separated in SDS-PAGE and transferred onto nitrocellulose membrane. After blocking with 5% skimmed milk (Marvel, Camlab, Cambridge, UK) in TBS (Santa Cruz)/0.1% Tween20 (Sigma) for 30 min at RT, the membranes were incubated with primary antibodies in 5% skimmed milk/TBS-T. The following primary antibodies were used for western blotting: EGFR (Cell Signaling), pEGFR (Abcam), Actin (Millipore).The blots were washed three times with TBS-T and then incubated with corresponding IRDye secondary antibodies 680RD or 800CW (Li-Cor, Cambridge, UK, 0.05 µg/ml). After three times washing with TBS-T protein signals were visualized using the ODYSSEY Sa infrared system (Li-Cor) and Image Studio software 2.0 (Li-Cor). Full scans of Western blots are shown in Supplementary Figure 5.