Western Blot Analysis of Lung Tissue

Western blot was performed as previously described [22 (link)]. Total proteins (30 ug) from lung tissue were separated by SDS-PAGE gels and then transferred to 0.22-µm PVDF membranes (Bio-Rad). After blocking, the membranes were incubated with primary antibodies against RhoA (1:1000 dilution; cat. no. 2117s; CST), ROCK1 (1:1000 dilution; ab156284; Abcam), MLC2 (1:1000 dilution; cat. no. 3672s; CST), p-MLC2 (1:1000 dilution; cat. no. 3671s; CST), 8-OHdG (1:500 dilution; sc-393,871; Santa Cruz), or α-Tubulin (1:1000 dilution; cat. no. 3873 S; CST) overnight at 4˚C, respectively; followed by the appropriate horseradish peroxidase-conjugated secondary antibodies (Dako). The expression was visualized using an enhanced chemiluminescence detection kit (Thermo Scientific). Semiquantitative analysis was done using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences). Additional file 1 is the original WB image in the manuscript.