Pab12124

Reagents were purchased or obtained from the following sources: rabbit antibody against Arg-II (#55003) was from Cell Signaling Technology (Danvers, MA, USA); mouse antibody against HIF1α (610958) was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); rabbit antibody against HIF2α (PAB12124) was from Abnova (Taipei, Taiwan); rabbit antibody against podocin (PA5-37284) was from Invitrogen/Thermo Fisher Scientific (Waltham, MA, USA); mouse antibody against synaptopodin (sc-515842) and mouse antibody against ACE1 (sc-23908) were from Santa Cruz Biotechnology (Dallas, TX, USA); mouse antibody against tubulin (T5168), mouse antibody against β-actin (A5441), dimethyloxaloylglycine (DMOG), and rotenone were from Sigma-Aldrich (St. Louis, MO, USA); IRDye 800-conjugated affinity purified goat anti-rabbit IgG F(c) was purchased from LI-COR Biosciences (Lincoln, NE, USA); goat anti-mouse IgG (H + L) secondary antibody Alexa Fluor^® 680 conjugate was from Invitrogen/Thermo Fisher Scientific (Waltham, MA, USA).

Isolated retinas were sonicated in 200 µl of 100 mM Tris/HCl (pH 8,0). After centrifugation (1000 × g; 3 min) protein concentrations were determined in the supernatants using Bradford reagent (BioRad, Hercules, CA, USA). Standard SDS-PAGE and Western blotting were performed using the following primary antibodies: rabbit anti-HIF1A (1:2000–1:4000, NB100-479, Novus Biologicals, Cambridge, UK); rabbit anti-HIF2A (1:1000, PAB12124, Abnova, Aachen, Germany); rabbit anti-pSTAT3_Tyr705 (1:500, #913L, Cell Signaling Technology, Danvers, MA, USA); rabbit anti-STAT3 (1:1000, D3Z2G, Cell Signaling Technology); mouse anti-GFAP (1:1000, G3893-Clone G-A-5, Sigma, Buchs, Switzerland); mouse anti-ACTB (1:10,000, A5441, Sigma). Primary antibodies were diluted in 5% non-fat blocking milk (BioRad, Cressier, Switzerland) in TBST, added to the membrane and incubated over night at 4 °C with gentle agitation. Appropriate HRP-conjugated secondary antibodies were added and signals detected using the Western lightning chemiluminescence reagent (PerkinElmer, Waltham, MA, USA). Signals were analysed using X-ray films.