66009 1 ig

According to our previous studies, total protein was extracted from retinal tissues or RGCs using lysis buffer (Zhang et al. 2017a (link), b (link)). Protein concentrations were measured using a NanoPhotometer N50 Touch spectrophotometer (IMPLEN, Heidelberg, Germany). Equal amounts of protein per lane (30 µg) were run on a 10% SDS–PAGE gel and then transferred to polyvinylidene difluoride membranes. The blots were probed with primary antibodies against caspase-3 ([1:1000], 9662; CST, Danvers, MA, US), cleaved caspase-3 ([1:1000], 9661; CST, Danvers, MA, US), Tle4 (15140, [1:1500]; Novusbio, Minneapolis, MN, US), and β-actin (66009-I-Ig, [1:1000]; Proteintech, Rosemont, IL, US) followed by incubation with secondary antibodies (goat anti-rabbit IgG (H + L) HRP [1:5000] or goat anti-mouse IgG (H + L) HRP [1:5000], Affinity, Cincinnati, OH, US). The expression of target proteins was normalized to the corresponding β-actin expression level in the same sample and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Each immunoblot was repeated three times to confirm the results.

Cells were lysed using RIPA reagent (Pierce, Thermo Scientific) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Scientific), and the cell lysates were denatured with loading buffer. For immunoblot analysis, immunoprecipitates or whole-cell lysates were loaded and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and the proteins were transferred onto nitrocellulose membranes (Millipore) for immunoblot analysis as described previously^{51 (link)}. The following antibodies were used for immunoblot analysis: Anti-STING (1:1000, #13647), anti-TBK1 (1:1000, #3504), anti-p-IRF3 (1:1000, #4947), anti-IRF3 (1:1000, #4302), anti-p-STAT1 (1:1000, #9167), anti-cGAS (1:1000, #31659), and anti-streptavidin-HRP (1:1000, #3999) were purchased from Cell Signaling Technology. Anti-NMT1 (1:2000, ab186123), anti-ICP5 (1:3000, ab6508), anti-LC3B (1:2000, ab192890), and anti-p-TBK1 (1:1000, ab109272) were purchased from Abcam. Anti-Myc (1:5000, M4439) and anti-Flag (1:5000, F1804) antibodies were purchased from Sigma. Anti-β-actin (1:20,000, 66009-I-Ig) and anti-ARF1(1:1000, 20226-1-AP) were purchased from Proteintech. Anti-HA (1:2000, TA180128) was purchased from Origene.