Superoxide dismutase 2 sod2

After herbal formula B401 or sham treatment, the four groups of ICR mice were anaesthetised with urethane (1.5 mg/kg); subsequently, transcardial perfusion with physiological saline was conducted. Uterine tissue specimens were homogenised in a buffer solution. Then, proteins in the separated solution were quantified using a BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and separated on 12.5% or 15% SDS polyacrylamide gels (Bionovas Pharmaceuticals Inc., Washington DC, USA). The separated proteins were transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Barrington, IL, USA). The antibodies used in this study were β-actin (Thermo Fisher Scientific Inc.) and superoxide dismutase 2 (SOD2) (Cell Signaling Technology Inc.), which were detected using suitable HRP-conjugated secondary antibody (Santa Cruz Biotechnology Inc.). Immunostaining was visualised using the enhanced chemiluminescence (ECL) substrate (Millipore, Billerica, MA, USA) and was quantified using ImageJ analysis software (version 1.48t, NIH, Wayne Rasband, Washington DC, USA).

The following primary antibodies were used for western blot analysis: TNF-α (1:500, 52B83; sc -52,746, Santa Cruz), total-nuclear factor-kappa B (NF-κB) (1:1000, #8242; Cell signaling Technology, Danvers, MA, USA), phospho-NF-κB (1:1000, #3033; Cell signaling Technology, Danvers, MA, USA), cyclooxygenase 2 (COX2) (1:1000, #4842S; Cell signaling Technology, Danvers, MA, USA), superoxide dismutase 2 (SOD2) (1:1000, 13141S; Cell signaling Technology, Danvers, MA, USA), heme oxygenase 1 (Hmox1) (1:1000, 70081S; Cell signaling Technology, Danvers, MA, USA), glutathione peroxidase (GPX) (1:1000, B-6; sc -133,160, Santa Cruz), Catalase (1:3000, H-9; sc271803, Santa Cruz), and α-tubulin (1:3000, #2144; Cell signaling Technology, Danvers, MA, USA).

The protein concentration was estimated, using the Bio‐Rad Bradford reagent. Equal amounts of total proteins (30 μg) were resolved, using SDS‐PAGE and transferred to polyvinylidene membranes. Membranes were probed with overnight primary antibody and 1 h of secondary antibody as described before (Veeranki et al. 2014). After probing the membranes with primary and secondary antibodies along with appropriate washes, chemiluminescence signal was detected using Bio‐Rad ChemiDoc™ XRS + System and Image Lab™ Software (Bio‐Rad, Hercules, CA). Antibodies: Connexin 43 is from Invitrogen (#355000; Carlsbad, CA); GAPDH is from Millipore (#374); Drp1 (#101270), Mfn2 (#100560), cluster of differentiation (CD31) (#1506), and Catalase (#50508) are from Santa Cruz biotech (Dallas, TX); superoxide dismutase 2 (SOD2) (#13141), Caspase3 (#9662) and pDrp1 (#6319) are from Cell Signaling Technologies (Danvers, MA); Tek (# 69753) is from Novus Biologicals and Bax (#182733; Littleton, CO) & Bcl2 (#692) are from Abcam (Cambridge, MA).