Ab270253

Liposomes (40 μL) were incubated with (1) BAX proteins (1 μM) treated with tBID (200 nM) for 15 min or (2) BCL-X_L proteins (1 μM) for 45 min in liposomal release assay buffer at room temperature. Protein-containing solutions were then added to a Sepharose CL-2B (3 mL, GE Healthcare) size exclusion column equilibrated with liposomal release assay buffer and 14 equivalent fractions (250 μL) were collected. Liposome-containing fractions were identified by adding 10% Triton X-100 to the fractions and measuring fluorescence associated with ANTS/DPX release. SDS PAGE followed by western blot analysis was conducted to identify BAX or BCL-X_L in the fractions using 2D2 mouse monoclonal BAX antibody (Santa Cruz Biotechnology Cat# sc-20067; RRID: AB_626726; 1:200) or mouse monoclonal anti-BCL-X_L (2H12) antibody (Abcam Cat# ab270253; RRID: AB_2536307; 1:1000), respectively.

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.