Analyzing Oxidative Stress Markers in MC3T3-E1 Cells

Whole cell extracts of MC3TE-E1 for western blotting were prepared after the indicated treatment for 3 days as previously described [19 (link)]. 20 μg protein from total cell lysate was prepared using PRO-PREPTM protein extraction solution (Boca Scientific Inc., Boca Raton, FL) and carried out subsequent electrophoresis, according to the manufacturer’s instruction. The primary antibodies against the following proteins: FoxO1 (Abcam, ab52857, 1:1000), SIRT1 (Abcam, ab189494, 1:1000), human catalase (CAT, Abcam, ab130029, 1:1000), glutathione peroxidase (GPX1, Abcam, ab108427, 1:1000), superoxide dismutase 2 (SOD2, Abcam, ab252426, 1:1000). Expression levels of the target protein were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Boster, Wuhan, China, 1:2000) levels in each sample. The following day, HRP-conjugated goat anti-rabbit, which used as secondary antibodies, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the results were detection and analysis by using an iBrightCL1000 (Invitrogen, Carlsbad, CA).