Ab190952

Protein was detached in RIPA buffer (Applygen) and quantitated by a protein quantitative kit (Beyotime). 20 μg/lane Protein was segregated by sodium dodecyl sulfate-polyacrylamide gel and then transferred on polyvinylidene fluoride membrane (Solarbio). The membrane was blockaded in 5% non-fat milk, and then nurtured with the antibody against BCL2L2 (ab190952, 1:2000, Abcam, Cambridge, MA, USA) overnight and a secondary antibody IgG labeled by horseradish peroxidase (HRP) (ab97200, 1:20,000, Abcam) for 2 h. GAPDH (ab245356, 1:5000, Abcam) was used as a loading control. After hatching with ECL reagent (Amyjet Scientific, Wuhan, China), the blots were visualized by ChemiDoc XRS+ system (Bio-rad Laboratories, Richmond, California, USA) and analyzed Image J v1.8 software (NIH, Bethesda, MD, USA).

A standard protocol of western blotting was performed. Briefly, cells were lysed using RIPA buffer, and total protein was extracted. After the concentration was determined, the protein was loaded with buffer onto SDS-PAGE gels with subsequent electrophoresis. The protein was then transferred to a PVDF membrane, which was blocked with 5% nonfat milk. Primary antibodies against BCL2L2 (BCL-w) (ab190952; 1:200; Abcam), BCL2L1 (BCL-xL) (ab270253; 1:200; Abcam), FGFRs (ab76464 for FGFR1 at 1:500; ab109372 for FGFR2 at 1:500; ab133644 for FGFR3 at 1:500; ab178396 for FGFR4 at 1:500; Abcam) and NCAM1 (ab9272; 1:500; Abcam) were applied overnight. The corresponding secondary antibodies and ECL were routinely applied. Densitometry was analyzed using ImageStudio software.