Anti smac

The total protein of lung tissues and cells was collected with RIPA buffer (Servicebio, Wuhan, China) with cocktail and phosphatase inhibitors (Thermo Fisher Scientific, Wuhan, China), followed by electrophoresed through 8–12% sodium dodecyl sulfate–polyacrylamide gel and then transferred to 0.45 μm PVDF membrane (Merck Millipore, USA). A total of 30 ug of protein was used for western blot experiments. After blocked with 5% milk, the membrane was immunoblotted with following primary antibodies(Except for labeled, all from Cell Signaling Technology, MA, USA) at 4 °C overnight: anti-ERRα (1:1000), anti-ZO-1 (1:1000; Abcam), anti-VE-cadherin (1:1000), anti-Occludin(1:1000), anti-JAM-A(1:1000; Abcam), anti-Sirt3 (1:1000), anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-Smac (1:1000), anti-Cytochrome c (1:1000), anti-caspase 3 (1:1000), anti-cleaved caspase 3 (1:500), anti-caspase 9 (1:1000), anti-cleaved caspase 9 (1:500), anti-LC3A/B(1:1000), anti-Beclin1 (1:1000), anti-p62 (1:1000), or anti-GAPDH (1:10000, Abcam, internal control). After incubated with the HRP-conjugated secondary antibodies at 25℃ for 1 h, the immunoblots were visualized using the ECL system. The gray value was analyzed using the Image J and calculate the relative protein level based on the density ratio of the target protein to GAPDH (internal control).