Glyceraldehyde 3 phosphate dehydrogenase

After transfection for 48 hours, total protein extracted from the CC was obtained. Protein samples were separated by electrophoresis in 10% sodium dodecylsulfate polyacrylamide gels and the protein was transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat dry milk in PBS for 2 hours. Samples were incubated overnight at 4°C with primary antibodies for heme oxygenase-1 (HO-1; 1:500; Abcam, Cambridge, MA, USA). The next day, membranes were washed three times, each time for 10 minutes with PBS containing 0.5% Tween 20, and then incubated with secondary antibodies (1:10,000; ZSGB, Beijing, China) for 1 hour at room temperature. Membranes were rinsed with PBS before scanning. Images were captured on the Gel Doc XR System (Bio-Rad, Richmond, CA, USA). Western blotting bands were quantified using Quantity One software (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase (1:1,000; ZSGB) served as an internal control.