Western blotting analysis of apoptosis markers

To further determine the effect of RAPA on the expression of apoptotic proteins, such as cleaved-PARP, Bax, and Bcl-2, fibroblasts were seeded in six-well plates at a density of 5 × 10⁵ and cultured in DMEM supplemented with 10 % foetal bovine serum and 1 % penicillin. When the cells were confluent, they were transfered into serum-free medium overnight and then pre-treated with or without different concentrations of RAPA for 48 h. Then, total proteins were extracted from cultured cells using radioimmuno-precipitation assay (RIPA) lysis buffer (Sigma-Aldrich, St Louis, MO, USA). Protein concentrations were determined using a bicinchoninic acid assay (BCA; ThermoFisher, MA, USA). Thirty micrograms of each protein lysate was added to each lane and resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, Sigma Aldrich, St Louis, MO, USA) on 12 or 6 % gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The primary antibodies were diluted in 1 % (w/v) skimmed milk powder in TBS-Tween and incubated overnight at 4 °C. Membranes were then washed and incubated with the appropriate secondary antibodies conjugated with IRDye 800CW (molecular weight = 1162 Da). Antibody reactivity was detected after exposure in an Odyssey infrared imaging system (LI-COR, Nebraska, USA).All the experiments were repeated three times.