Anti sod1

HLECs from HDACi-treated groups and controls were used to quantitatively detect SOD1 using a commercially available ELISA (AB Frontier). Absorbance was assessed at 450 nm using the 680XR Microplate reader (Biorad, Hercules, USA). For the SOD1 ELISA, primary antibody was a rabbit monoclonal anti-SOD1 (Sigma) antibody and the secondary antibody was a goat anti-rabbit antibody. The analysis of SOD1 levels was performed by the kit manufacturer. The SOD1 standard curve was generated by linear regression analysis in each group.

All chemicals used in study were of analytical grade and were either procured from Indian manufacturer (SRL India, HiMedia chemicals) or obtained from Sigma Aldrich (St Louis, MO), Thermo Fisher Scientific Inc. (USA) etc. Roswell Park Memorial Institute (RPMI-1640) medium, High glucose Dulbecco’s Modified Eagle’s medium (DMEM), Eagle’s Minimum Essential Medium (EMEM), penicillin, streptomycin, blasticidin, trypsin, mannan, rotenone, antimycin A, bovine serum albumin (BSA), ethidium bromide, sodium pyruvate, uridine, protease and phosphatase inhibitor cocktails, REDTaq ReadyMix PCR reaction mix, antibodies (anti MnSOD, anti-SOD1, phospho JNK, phospho p38, anti rabbit/ mouse-HRP, anti β-actin-HRP) etc. were obtained from either Sigma Aldrich (St Louis, MO) or Cell Signaling technology, USA, whereas 3, 3’-DihexyloxacarbocyanineIodide [DiOC₆ (3)], JC-1, 5-(and-6)-chlormethyl 2′,7’dichlorodihydrofluorescein diacetate acetyl ester [CM-H₂DCFDA], 10-N-nony1–3, 6-bis [dimethylamino] acridine (NAO), Dihydroethidine (DHE), sulphorhodamine-B (SRB), fetal bovine serum (FBS), Keratinocyte-SFM (1X) were procured from Thermo Fisher Scientific Inc. (USA).

The protein lysates were prepared with protease inhibitors cocktail‐contained radioimmunoprecipitation assay lysate (Beyotime). The protein samples were boiled at 98°C for 10 min. Next, 60 μg of proteins was used for analysis on 10% sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (SDS‐PAGE; Bio‐Rad, Hercules, CA). The SDS‐PAGE gels were transferred to 0.45 μm polyvinylidene difluoride (PVDF) membranes (Bio‐Rad) for 45 min. Then, the protein‐carried PVDF membranes were blocked with 2% bovine serum albumin, and they were incubated with the indicated primary antibody overnight. Afterward, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody for 45 min diluted at 1:50,000 (Bioworld Technology, Nanjing, China). Importantly, the primary antibodies including anti‐GAPDH, anti‐FBXW7, anti‐Bax, anti‐Bcl‐2, anti‐TNF‐α, anti‐IL‐1β, anti‐IL‐6, anti‐CAT, anti‐GSH, and anti‐SOD1 were purchased from Sigma‐Aldrich.