B8429

Lung tissues or MLE-15 cells were lysed with RIPA containing protease inhibitors to obtain the total protein (Thermo Fisher Scientific). The protein was separated by 10% SDS-PAGE and then transferred to a PVDF membrane. The primary antibodies against JNK (1:1000, #9252; Cell Signaling Technology, Danvers, MA,USA), p-JNK (1:1000, #9255; Cell Signaling Technology), Bcl-2 (1:1000, SAB4500003; Sigma Aldrich), Bax (1:1000, B8429; Sigma Aldrich), and Bak (1:1000, SAB4502532; Sigma Aldrich) were incubated with the membrane at 4℃ overnight. Thereafter, the membranes were incubated with secondary antibody (1:5000, Sigma Aldrich) at room temperature for 40 min. The bands were visualized with ECL detection reagents (GE Healthcare, Little Chalfont, Buckinghamshire, UK). GAPDH served as the internal reference, and the ratio of the gray value of target band to internal reference band was considered as the relative protein level. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the gray value.