Ecl working solution

Cells were lysed with enhanced radio immunoprecipitation assay lysis appended to protease inhibitor (BOSTER Biological Technology Co., Ltd., Wuhan, Hubei, China). The protein concentration was measured by the Bicinchoninic Acid Assay Kit (BOSTER). Following electrophoresis separation, the protein was transferred onto polyvinylidene difuoride membrane. After blocked with 5% bovine serum albumin for 2 h to inhibit non-specific binding, membranes were incubated with diluted primary antibodies (Supplementary Table 3) overnight at 4 °C. The membranes were then incubated with the horseradish peroxidase (HRP)-labeled secondary antibody (1: 2000) (Supplementary Table 3) at room temperature for 1 h. The membrane was developed with ECL working solution (EMD Millipore) for 1 min with the results analyzed with ImageJ software.

RB tissues and normal retinal tissues or cells were lysed to isolate total protein. Proteins were separated by 10% sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrotransferred to nitrocellulose membrane (Millipore, Temecula, CA, USA) and blocked with 5% BSA at room temperature for 2 h to block nonspecific binding. Primary rabbit anti-p38 MAPK, anti-P-p38 MAPK, USP22, SIRT1, SOST, and GAPDH (ab128915; 1: 10000, Abcam) were incubated with the membrane overnight at 4°C. The membrane was then incubated with HRP-labeled goat anti-rabbit secondary antibody (ab6702; 1:2000, Abcam) for 1 h at room temperature, developed using ECL working solution (EMD Millipore, USA) and exposed. Image J analysis software was used to quantify the gray scale of each band in the Western blot Image. GAPDH was taken as internal reference.

Cells were collected after trypsin digestion and lysed with enhanced RIPA buffer containing protease inhibitor (Boshide Co., Ltd., Wuhan, China), and protein concentration was determined by a BCA Protein Assay Kit (Boshide Co., Ltd., Wuhan, China). Equal amounts of protein were separated by 10% SDS-PAGE and transferred to a PVDF membrane, followed by blockade of nonspecific binding with 5% BSA at room temperature for 2 hours. Subsequently, membranes were incubated at 4°C overnight with primary rabbit antibodies to HDAC3 (Ab32369, 1 : 2000, Abcam), LC3B (ab51520, 1 : 2000, Abcam), ULK1 (ab203207, 1 : 2000, Abcam), Beclin-1 (ab207612, 1 : 1000, Abcam), and Ac-FOXO1 (AF2305, 1 : 1000; Affinity Biosciences, Jiangsu, China). The membranes were the incubated with HRP-conjugated goat anti-rabbit secondary antibody (ab205719, 1 : 2000, Abcam) at room temperature for 1 hour. ECL working solution (EMD Millipore, USA) was applied for 1 minute at room temperature to visualize membranes. ImageJ was used to quantify the gray levels of each band, with GADPH (rabbit, ab181602, 1 : 10000) as the loading control. The experiment was repeated three times.

First, we lysed the samples digested and collected from tumor tissues of each group with enhanced RIPA Lysis Buffer containing protease inhibitors (Wuhan Boster Biological Technology). The protein concentration was measured using a BCA protein assay kit (Wuhan Boster Biological Technology) to ensure equal protein loading. Subsequently, protein samples were separated via SDS-PAGE and then electro-transferred onto PVDF membranes. On the PVDF membranes, we blocked with 5% BSA at room temperature for 1 h to prevent non-specific binding, then added diluted primary antibodies, including β-actin (ab8226, 1/5000, Abcam), NDUFAF6 (ab110244, 1/1000, Abcam), NRF2 (SAB4501984, 1/1000, Sigma), and PD-L1 (ab213480, 1/1000, Abcam) and incubated overnight at 4 °C for specific protein binding. Afterward, the PVDF membrane was washed three times with PBST for 5 min each, then incubated with Anti-Mouse-HRP secondary antibody (Cat # 7076, 1/5000, CST) or Anti-Rabbit-HRP secondary antibody (Cat # 7074, 1/5000, CST) at room temperature for 1 h. Finally, an appropriate amount of ECL working solution (EMD Millipore, USA) was added to the transferred membrane and incubated at room temperature for 1 min. Excess ECL reagent was removed, sealed with plastic wrap, placed in a dark box, and exposed to X-ray film for 5–10 min, followed by development and fixation.