Secondary horseradish conjugated antibodies

Immunoblotting was performed to assess protein expression in response to the different treatments, as previously described [31 (link)]. Briefly, control and treated HuREC were lysed using radioimmunoprecipitation assay buffer lysis buffer containing 1% phosphatase and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Fifty micrograms of each protein sample was electrophoresed on SDS-PAGE and transferred onto a Polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked using 5% skim milk and incubated with the following primary antibodies: SIRT1 (1:1000; Cell Signaling) and p16^INK4a, TrxR2 (1:1000; Abcam, Cambridge, MA), p21^Waf1 and SOD2 (1:500; Santa Cruz Biotech, Dallas, TX, USA), and corresponding secondary horseradish-conjugated antibodies (GE Healthcare, Pittsburg, PA, USA). After immunoblotting, the membranes were stripped using stripping buffer (Bio-Rad) and re-probed with anti-β-actin antibody (1:3000; Sigma-Aldrich, St. Louis, MO, USA). Chemiluminescence-based assay was used for band detection (ThermoFisher, Waltham, MA, USA). Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/).

Proteins were extracted from retinas and HREC as previously described [8 (link)]. The extracted proteins were quantified by using BioRad Protein DC Assay (Bio-Rad). Western blotting analysis was carried out as described [8 (link)] using specific antibodies for STAT3 phospho tyrosine 705 (pSTAT3; 1:2000; Cell Signaling, Danvers, MA) and TIMP3 (1:1000; Cell Signaling Technology, Beverly, MA), and corresponding secondary horseradish-conjugated antibodies (GE Healthcare, Pittsburg, PA). Phospho-STAT3 levels were normalized to total STAT3 (1:1000; Cell Signaling). Actin antibody was used as an internal control for TIMP3 expression (1:1000; Santa-Cruz Biotech). Chemiluminescence-based assay was used for protein detection (ThermoFisher, Rockford, IL).