Quantitative Western Blot Analysis

Western blotting was performed as previously described 18 (link), 21 (link). Briefly, total protein from each group of cells was subjected to SDS-PAGE on a 12% denaturing gel. Afterwards, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). After blocking and washing the membrane, primary antibodies (Table 1) were added and the membrane was incubated at 37 °C for 15 min. After extensive washing, secondary antibodies (Table 1) were added and incubated at 37 °C for 45 min. The membrane was subjected to four 14 min washes with Tris-buffered saline-Tween 20 (TBST) at room temperature. The membrane was then developed using an enhanced chemiluminescence (ECL) kit (Pierce Biotechnology, MA, USA) and exposed to X-ray film (Sigma-Aldrich) for visualization. The gray levels of western blotting protein band were quantified by using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA). To determine relative expression levels of target proteins, the formula was used as follows: (Experiment group_target protein gray level value/Experiment group_GAPDH protein gray level)/(Control group_target protein gray level value/Control group_GAPDH protein gray level) value. The protein levels were calibrated based on levels of GAPDH.