Anti rhodopsin 1d4

Western blot analysis was performed on retinae, which were harvested. Samples were lysed in hypotonic buffer (10 mM Tris-HCl [pH 7.5], 10 mM NaCl, 1,5 mM MgCl₂, 1% CHAPS, 1 mM PMSF, and protease inhibitors) and 20 µg of these lysates were separated by 12% SDS-PAGE. After the blots were obtained, specific proteins were labeled with anti-1D4 antibody anti-Rhodopsin-1D4 (1:1000; Abcam, Cambridge, MA), and anti-β-tubulin (1:10000; Sigma-Aldrich, Milan, Italy) antibodies.

Lentiviral vectors carrying short hairpin RNA (shRNA) targeting ADAM33 (LV-ADAM33) and negative control vectors were obtained from Shanghai HanBio Technology (China). The infection enhancers HitransG A and HitransG P were obtained from Jikai Gene Company (China). Enzyme-linked immunosorbent assay (ELISA) kits for interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 17 (IL-17) were purchased from Hangzhou Kelian Biological Company (China). Anti-PI3 kinase p85 alpha, antirhodopsin [1D4], and anticyclin D1-C-terminal [EPR2241] antibodies were obtained from Abcam (UK). Anti-AKT (pan) (C67E7), antiphospho-AKT (Ser473), anti-mTOR (7C10), antiphospho-mTOR (Ser2448), anti-ROCK1 (C8F7), and antiphospho-forkhead box protein (FOXO1) (C29H4) antibodies were purchased from Cell Signaling Technology Corporation (USA).

20 μg/ml of K-874A was immobilized on Pierce Nickle Coated Plates, Clear, 96-well (Thermo Fisher Scientific). PBST was used for washing after each step. K-874A-coated wells were blocked with 5% skim milk in PBST for 1 hr. Recombinant S proteins produced by HEK293 cells were purchased from BioServ. Each S protein was diluted 5, 50, or 500 ng/ml and 100 μl of diluted S protein was incubated for 1 hr. The S protein captured by K-874A was detected with anti-rhodopsin [1D4] (Abcam) mouse monoclonal antibody and anti-mouse IgG goat polyclonal antibody-HRP (Abcam). The o-phenylenediamine dihydrochloride substrate tablet (Thermo Fisher Scientific) dissolved with Stable Peroxide Substrate Buffer (Thermo Fisher Scientific) was used as a substrate. The absorbance at 450 nm was measured with GloMax Explorer System (Promega).