Anti rhodopsin

After being anesthetized by 1% pentobarbital (150 mL/kg), RCS rats were perfused with normal saline and 4% paraformaldehyde via the circulation system on 3, 6, 9 and 12 post-operation weeks as we previously described^{40 (link)}. Eyeballs were enucleated and fixed in 4% paraformaldehyde for 3 hours. After incubation in 30% glucose solution overnight, retinal tissues were collected and serially frozen-sectioned to a thickness of 10 μm. Immunofluorescence was performed as previously described^{57 (link)}. In detail, sections that crossed the optic disc were rinsed in 0.1 M PBS and blocked for 30 min in 10% of goat serum diluted in 0.1% of Triton X-100. Then, sections were incubated with the primary antibodies, anti-human mitochondrial antibody (1:200, mouse, Abcam), anti-human mitochondrial antibody (1:200, rabbit, Millipore, Billerica, MA), anti-recoverin (1:1000, rabbit, Millipore), anti-rhodopsin (1:8000; rabbit, Sigma-Aldrich), anti-Iba1 (1:500; Wako, Japan) and GFAP (1:500, rabbit, Sigma Chemical Co) in 1% BSA at 4 °C overnight. Secondary antibodies, cy3-or 488-conjugated (Invitrogen), were then implemented (1:400, 37 °C, 2 h). Some sections were processed only with the secondary antibodies as negative controls. Before examination with a confocal laser scanning microscope (Leica, Germany), sections were counterstained with DAPI (Sigma Aldrich, St. Louis, MO, USA).

Anti-human TRPM1 (F-3, western blotting 1: 250; immunoprecipitation 1:50; immunohistochemistry 1:100), anti-HSP70 (W27, for western blotting, 1:1000), anti-HSP90 α/β (F-8, for western blotting, 1:1000), anti-CDC37 (C-11, for western blotting, 1:1000) antibodies, and anti-HSP90 α/β conjugated agarose were from Santa Cruz Biotechnology. Anti-mouse Trpm1 (western blotting 1:200; immunohistochemistry 1:100; immunofluorescence 1:100) was from Novus Biologicals. Anti-cleaved caspase 3 (Asp175) (western blotting 1:1000; immunohistochemistry 1:100), anti-AKT (western blotting 1:1000; immunohistochemistry 1:100), anti-phospho-AKT(Ser473) (D9E, immunohistochemistry 1:100), anti-β-Actin (13E5, western blotting 1:2000; immunofluorescence 1:500), anti-PARP1 (# 9542, western blotting 1:1000) and anti-Ki67 (D2H10, immunohistochemistry 1:100) antibodies were purchased from Cell Signaling Technology. Anti-Rhodopsin (Rho 1D4, immunohistochemistry 1:100), anti-RPE65 (immunofluorescence 1:250), anti-FLAG M2 (western blotting 1:1000) antibodies and anti-FLAG M2 affinity agarose gel were from Sigma-Aldrich. Anti-GFAP antibody (immunohistochemistry 1:100) was from Dako. Biotinylated Peanut Agglutinin ((immunohistochemistry 1:500) was obtained from Vector Laboratories. AUY922 and MG132 were from MedChem Express.

Total protein from cells or zebrafish larvae (n = 15 per genotype), was extracted using the M-PER protein lysis buffer (ThermoScientific, Beverly, MA) containing protease inhibitors (Roche, Indianapolis, IN, USA). Approximately 25 μg of total protein was then electrophoresed on 4–12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were probed with primary antibodies against β-Actin or Gapdh (1:10,000, Sigma/Millipore, Cambridge, MA, USA), anti-V5 (1:1000, Sigma/Millipore, Cambridge, MA, USA), anti-Lrat (1:1000; Abcam, Cambridge, MA, USA), anti-Rhodopsin (1:1000, Sigma/Millipore, Cambridge, MA, USA), anti-PARP1 (1:2000; Cell Signaling, Danvers, MA) and anti-Red/Green cone opsin (1:1000, Sigma/Millipore, Cambridge, MA, USA) in antibody buffer (0.2% Triton X-100, 2% BSA, 1X PBS) [33 (link),40 (link),48 (link),49 (link)]. HRP conjugated secondary antibodies (BioRad, Hercules, CA, USA) were used at 1:10,000 dilution. Protein expression was detected using a LI-COR Odyessy system and relative intensities of each band were quantified (densitometry) using Image J Software version 1.49, and normalized to the loading control.