Anti c met 25869 1 ap

We used 1 mL RIPA buffer (Beyotime, China) to extract the total proteins of cells at 0°C for two hours, and centrifuged at 4°C, 15 000 r/minute. We collected the supernatant, trimmed the protein system and boiled for five minutes with loading buffer (Beyotime, China). Then, 10% SDS‐PAGE electrophoresis was used to separate proteins with different molecular weights and transferred to PVDF membrane (0.45 μm, Thermo Scientific), and 10% skimmed milk sealed the membranes for two hours. Diluting the primary antibody to the working concentration: anti‐CAPN1 (10538‐1‐AP, proteintech, USA) 1:500; anti‐PTPN1 (11334‐1‐AP, proteintech, USA) 1:1000; anti‐c‐Met (25869‐1‐AP, proteintech, USA) 1:500; PIK3R2(60225‐1‐Ig, proteintech, USA) 1:3000; antiphospho Met (ab73992, abcam, USA) 1:1000; anti‐phospho PIK3R2 (17366S, Cell Signaling Technology, USA) 1:1000. Second antibody incubated membranes for two hours at ordinary temperature. ECL western blotting substrate (Thermo Fisher, USA) was used for chemiluminescence. GelDoc XR+ instrument (Bio‐Rad, USA) was used to collect the luminescent signals.

The following antibodies were purchased: anti-BRD4 (E2A7X) from Cell Signaling Technology, anti-BRD2 (D89B4) from Cell Signaling Technology, anti-BRD3 (2088C30) from Abcam Technology, anti-c-Myc (A19032) from ABclonal Technology, anti-c-Met (25869-1-AP) from Proteintech Technology, anti-EGFR (2232) from Cell Signaling Technology, and anti-GAPDH (AP0063) from Bioworld Technology Inc. All antibodies were utilized at 1:1000 dilutions, except GAPDH (1:10,000). JQ1 (HY-13030) and (2-Hydroxypropyl)-β-cyclodextrin (HY-101103) were purchased from Haoyuan Chemexpress Co., Ltd. Trichostatin A (T6270) was purchased from Target Mol, Inc. Vorinostat (T1583) was purchased from Target Mol, Inc. Osimertinib (HY-15772) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Reagents were dissolved in DMSO at 20 mmol/L, stored at -20℃, and diluted just before use. Lipofectamine 2000® transfection reagent (11668-019) was purchased from Life Technologies Co. Invitrogen (Carlsbad, CA, USA).