Goat anti mouse igg secondary antibody

Immunoblotting was performed as described previously^{49 (link),50 (link)}. Briefly, cell pellets were lysed using RIPA lysis buffer (Millipore, #20-188), and the protein concentration was determined by a Bradford assay (Bio-Rad, #500-0006) using a NanoDrop 2000 (Thermo Fisher Scientific). For immunoblot analysis, 15-30 μg of protein was used along with antibodies against phospho-Rb ([S780] Cell Signaling Technology, #9307, 1:1,000), RB1 (Cell Signaling Technology, #9309, 1:1,000), CDK1 (Cell Signaling Technology, #9116, 1:1,000), ACSL4 (Santa Cruz Biotechnology, #sc-271800, 1:1,000), GPX4 (R&D Systems, #MAB5457, 1:1,000), SLC7A11 (Cell Signaling Technology, #12691, 1:1,000), FSP1 (Santa Cruz Biotechnology, #sc-377120, 1:300), DHODH (Proteintech, #14877-1-AP, 1:1,000), DGAT1 (Santa Cruz Biotechnology, #sc-271934, 1:300), vinculin (Sigma, #V4505, 1:50,000), and tubulin (Sigma, #T9026, 1:10,000), Goat anti-rabbit IgG secondary antibody (Thermo Scientific, #31460, 1:5,000), Goat anti-mouse IgG secondary antibody (Proteintech, #SA00001-1, 1:5,000). The uncropped scans of the immunoblots used in this study are shown in the Source Data file.

The protein samples were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresed proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany), then blocked with 5% bovine serum albumin (BSA, Gbcbio, China). Membranes were probed with anti-p-H2AX (CST, United States), anti-H2AX (CST, United States), anti-p-SMC1 (Abcam, United Kingdom), anti-SMC1 (CST, USA), or anti-βactin antibodies (Proteintech, China), followed by a goat anti-rabbit IgG secondary antibody (#SA00001-2, Proteintech, China) or goat anti-mouse IgG secondary antibody (#SA00001-1, Proteintech, China) for 1 h. The blots were visualized using an ECL chemiluminescence substrate kit (#WBKLS0100, Millipore, United States).

The retina protein was extracted using RIPA buffer (P0013B, Beyotime, China) with PMSF and phosphatase inhibitors. The concentration of extracted protein was measured with BCA protein concentration assay kit (P0009, Beyotime, China). According to the measured value, the samples were adjusted to the same protein concentration (0.8 μg/μL) and denatured. 15 μL of each sample containing equal amounts of protein (12 μg) was taken for electrophoresis and transferred. The PVDF membrane with Western blotting was blocked with blocking solution (5% skimmed milk powder prepared with TBST) for 2 h, and then western blotting was incubated with primary antibody mTOR, 1:1,000 (Abcam), p-mTOR, 1:1,000 (Abcam), Rhodopsin, 1:500 (Abcam), and GAPDH, 1:10,000 (Prteintech) overnight at 4°C. After overnight, the membrane was washed three times with TBST and then incubated with goat anti-mouse IgG secondary antibodies 1:10,000 (Proteintech) and goat anti-rabbit IgG secondary antibodies 1:10,000 (Proteintech). After washed three times with TBST, the PVDF membrane was added with the ECL droplet to make it uniformly distribute on the surface of the whole membrane and was put into the gel imaging system to take photos.