Ab2918

Mouse myoblast C2C12 cells (ATCC) (2.0 × 10⁵ cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS, Gibco), penicillin (100 units/ml), streptomycin (100 mg/ml), and amphotericin B (250 ng/ml), and incubated in a 5% CO₂ humidified chamber at 37 °C. The cells were then incubated in FBS-free DMEM containing reagent (1 μM) at 37 °C for 120 min. As control experiments, the labelling was conducted in the presence of Rapamycin (10 μM). For western blot analysis, after washing twice with PBS, the cells were lysed with RIPA buffer (pH 7.4, 25 mM Tris–HCl, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.25% deoxycholic acid) containing 1% protease inhibitor cocktail set III (Calbiochem). The lysed samples were collected and centrifuged (15 200 × g, 10 min at 4 °C). The supernatants were mixed with 1/4 volume of 5× sample buffer (pH 6.8, 312.5 mM Tris–HCl, 25% sucrose, 10% SDS, 0.025% bromophenol blue) containing 250 mM DTT and incubated for 1 h at 25 °C. The samples were subjected to SDS-PAGE and electro-transferred onto an Immun-Blot PVDF membrane (Bio-Rad). The labelled FKBP12 was detected by chemiluminescence analysis using Streptavidin-HRP conjugate, rabbit anti-FKBP12 antibody (Abcam, ab2918, 1:1000) and anti-rabbit IgG-HRP conjugate (CST, #7074 S, 1:5000).