Rabbit anti β actin antibody

Retinal protein extraction, SDS-PAGE and an immunoblot analysis were performed as described previously.22 (link) Briefly, membranes were incubated in a blocking buffer containing rabbit anti-α-fodrin antibody (Abcam 1:2000) at room temperature for 1 h. The membranes were then incubated with HRP-conjugated anti-rabbit IgG (dilution 1:5000; Sigma), immunoreactive bands were developed with ECL prime (GE Healthcare, Life Sciences) and the bands were examined with ChemiDoc XRS+ (Bio-rad). As an internal control, membranes were incubated with rabbit anti-β-actin antibody (dilution 1:1000; Sigma) at 4° C overnight. The density of the immunoreactive bands was then determined with a digital scanner and Image J software.

After transfection for 48 hours, cells were lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich, St. Louis, MO). Then an equal amount of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred to a PVDF membrane with protein, which was blocked for 2 hours with 5% nonfat milk in TBST. Following incubation with the primary antibodies (a rabbit anti-LC3 monoclonal antibody, a mouse anti-TLR3 monoclonal antibody [Abcam, Cambridge, UK], and a mouse anti-TRIF polyclonal antibody [Sigma-Aldrich], all diluted at 1:500 dilution; a rabbit anti–β-actin antibody [Sigma-Aldrich], at 1:2,000 dilution) overnight at 4°C, membranes were washed in TBST for 5 minutes×3 times, followed by incubation with peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Sigma-Aldrich) at 1:2,500 dilution for 1 hour at room temperature, and developed using a chemiluminescence system (Pierce, Rockford, IL). The film was scanned and the density of the bands measured using Image Quant software (Molecular Dynamics, Sunnyvale, CA) and then expressed as the percentage of the density of the β-actin band.

Metformin (1,1-dimethyl biguanide hydrochloride) was purchased from Sigma (#D150959, Sigma-Aldrich, St. Louis, MO, USA) and dissolved in a serum-free medium to a stock solution of 1 M. Other chemicals and reagents were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and Merck (Merck KGaA, Darmstadt, Germany).
Antibodies were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and CST (Cell Signaling Technology, Beverly, MA, USA). The antibodies used for Western blots were anti-rabbit IgG-peroxidase (#A0545, Sigma), rabbit anti-GAPDH antibody (#G9545, Sigma), rabbit anti-β-actin antibody (#A2066, Sigma), anti-PARP antibody (#9542, CST), anti-caspase-3 antibody (#9662, CST) and anti-cleaved caspase-3 antibody (#9664, CST).