Sa00001 15

The total protein was extracted utilizing RIPA buffer (Solarbio), separated via SDS-PAGE gel (10%) and transferred to PVDF membrane (Beyotime). Subsequently, 5% slim milk was used to block membranes under indoor environment for 1 h, followed by reaction with primary antibodies against B-cell lymphoma-2 (Bcl2, 26 kDa, ab182858, 1:1000, Abcam), Bcl-2-associated X protein (Bax, 21 kDa, ab32503, 1:1000, Abcam), IL-6 (23 kDa, ab208113, 1:1000, Abcam), IL-1β (31 kDa, ab283818, 1:1000, Abcam), TNF-α (26 kDa, ab6671, 1:1000, Abcam), IL-10 (20 kDa, ab33471, 1:1000, Abcam), medullary differentiation protein 88 (Myd88, 33 kDa, ab133739, 1:5000, Abcam), Phospho-nuclear factor kappaB (NF-kB) p65 (p-p65, 65 kDa, 3033T, 1:1000, Cell Signaling Technology, Danvers, Massachusetts, USA), NF-kB inhibitor alpha (IκB-α, 36 kDa, AI096, 1:1000, Beyotime), Cleaved-caspase9 (35 kDa, 9505T, 1:1000, Cell Signaling Technology), and internal reference GAPDH (36 kDa, ab181602, 1:10000, Abcam) overnight at 4℃. Subsequently, Rabbit IgG H&L (HRP) secondary antibody (ab205718, 1:10000, Abcam) or Rat IgG(H + L) (HRP) secondary antibody (SA00001-15, 1:2000, proteintech, Wuhan, China) was applied to interact with the membrane for 2 h in indoor environment. Thereafter, BeyoECL Star Kit (Beyotime) was used to visualize the immunoblots.

The mouse neuroretinas were harvested and transferred to a tube containing RIPA buffer with protease inhibitors, homogenized with a motor grinder (VCX130, Sonics), and centrifuged for 10 min at 4 °C. The resulting supernatant was mixed with loading buffer (WB2001, NCM Biotech) and heated at 95 °C for 15 min. Each sample was then separated by SDS-PAGE and transferred onto PVDF membranes (catalog ISEQ00010, Millipore), followed by one hour of blocking in 5% nonfat milk in Tris-buffered saline (TBS) containing Tween-20 (TBS-T) at room temperature. Subsequently, the membranes were incubated with primary antibodies at 4 °C overnight, which included rabbit anti-PDE6β antibody (1:1000; catalog PA1-722; Thermo Fisher), rat anti-HA antibody (1:1000, catalog 11867431001, Roche) and rabbit anti-β-actin antibody (1:1000, catalog 3779, ProSci). After overnight incubation, the membranes were washed with TBS-T three times for 10 min each and then incubated with secondary antibodies for 1 h at room temperature, which included HRP-conjugated AffiniPure goat anti-rat IgG (H + L) antibody (1:5000, catalog SA00001-15, Proteintech) and HRP-conjugated AffiniPure goat anti-rabbit IgG (H + L) antibody (1:5000, catalog SA00001-2, Proteintech). After washing with TBS-T three times for 10 min each, the membranes were scanned using an Amersham Imager 600 (GE Healthcare, Freiburg, Germany).