Voltage dependent anion channel

Proteins (40 μg per lane) were separated with SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After being blocked with 5% skim milk in TBST for 1 h, the membranes were incubated with primary antibody against Dynamin-related protein 1 (DRP1; 1:1,000; Abcam), Mitofusin 2 (MFN2; 1:1,000; Abcam), NLRP3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), caspase-1 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), GSDMD (1:500; Abcam), interleukin-1β (IL-1β; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-18 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), synapsin 1 (1:2,500; Millipore, Billerica, MA, USA), PSD-95 (1:1,500; Abcam), voltage-dependent anion channel (VDAC; 1:1,000; Cell Signaling Technology, Danvers, MA, USA), or GAPDH (1:1,000; Cell Signaling Technology, Danvers, MA, USA). After three washes, the membranes were treated with species-specific peroxidase-conjugated secondary antibodies for 1 h at room temperature. Bands were visualized by enhanced chemiluminescence and quantified with Image Quant Software (Syngene).

Cell pellets were collected and lysed in RIPA buffer. Protein concentrations were determined with the Bicinchoninic acid assay using albumin as the standard. Proteins were resolved on SDS-PAGE gels (made with 2,2,2-Trichloroethanol). Before transferring proteins to low-fluorescence Polyvinylidene difluoride (PVDF) membranes using BioRad Turbo Blot, gels were activated with UV light. After transfer, membranes were imaged to allow for normalization to protein load. Membranes were incubated overnight with primary antibodies, Total OXPHOS Human WB cocktail (Abcam), Voltage-dependent Anion Channel (VDAC) (Cell Signaling; Danvers, MA, USA), or Cytochrome Oxidase IV (COX IV) (Cell Signaling). Secondary antibodies conjugated to horseradish peroxidase along with the Chemiluminescence kit (ThermoFisher) were used to visualize the immune reactions. Images were taken using a BioRad ChemiDoc XRS. Densitometry was performed using Image Lab software (BioRad, Hercules, CA, USA). Intensity of bands were normalized for protein load and to a standard run on each blot. Fold-change values were calculated from the average for No AMD or CFH low risk values.