Zb 5301

Total protein was extracted from cells or retina tissues utilizing RIPA buffer (Solarbio, Beijing, China). RIPA buffer (Solarbio, Beijing, China) was used to lyse cells or retina tissues. The protein concentration was determined using the BCA protein assay kit (Pierce, Appleton, WI, USA). Then, protein was separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by transference onto polyvinylidene difluoride (PVDF; EMD Millipore, Burlington, MA, USA) membranes. The membranes were blocked with 5% skim milk for 1 h. Then, the membranes were incubated with primary antibodies including anti-p53 (1:500, ab131442; Abcam, Waltham, MA, USA), anti-Bcl-2 (1:1,000, ab182858; Abcam, Waltham, MA, USA), anti-Bax (1:1,000, ab32503; Abcam, Waltham, MA, USA), anti-Cleaved-Caspase-3 (1:500, 33199M; BSM, Shanghai, China) and anti-β-actin (1:2,000, 20536-1-AP; Proteintech, Wuhan, China) overnight at 4 °C, followed by secondary antibodies (1:6,000, ZB-5301; ZSGB-BIO, Beijing, China) for 30 min at room temperature. The proteins were visualized using an ECL kit (KeyGen Biotech Co., Ltd., Jiangsu, China). β-actin was used as an internal control. The expression of protein was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

1 μg/mL His-Mb immunogen protein was coated in the 96-well enzyme label plate (NEST Biotechnology Co., Ltd., Wuxi, China) at 4 °C overnight. Then 300 μL 1% BSA (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China #A8010) sealing solution was added and incubated at 37 °C for 1 h. After that, added 200 μL test serum with a dilution ratio of 1:250, 1:1000, 1: 4000, 1:16,000, 1:64,000, and 1:256,000. The serum before immunization was used as the negative control and incubated at 37 °C for 1 h. 100 μL HRP-coupled secondary antibody (l:8000, ZSGB-BIO, #ZB-5301) was added and incubated at 37 °C for 1 h. Finally, added 100 μL TMB substrate and incubated at 37 °C in a dark place for 10–20 min, and then added 50 μL HCL to stop the reaction. The absorbance value of each hole at the wavelength of 450 nm (OD450) was detected by an enzyme labeling instrument (Hangzhou Allsheng Instruments Co., Ltd., Hangzhou, China, FlexA-200).

IP for His-Mb: Purified His-Mb protein was incubated with protein A magnetic beads and mouse anti-6 × His tag monoclonal antibody (Abcam, #ab18184)/mouse anti-IgG antibody (Abcam, #ab190475) overnight with shaking at 4 °C. The magnet was used to adsorb the beads, and the sample was washed three times with a binding buffer. Then, WB analysis was performed using rabbit anti-Mb monoclonal antibodies (1:2000, Abcam, #ab77232) and HRP-coupled goat anti-rabbit IgG secondary antibody (l:5000, ZSGB-BIO, #ZB-5301).
IP for Anti-Mb antibody: Total proteins from rat and mouse muscle tissue were extracted with IP lysis buffer and incubated with protein A magnetic beads and rabbit anti-Mb polyclonal/monoclonal antibody/rabbit anti-IgG antibody (Abcam, #ab172730) overnight with shaking at 4 °C. The magnet was used to adsorb the beads, and the sample was washed three times in a binding buffer. Moreover, WB analysis was performed using mouse anti-Mb antibody (1:100, Santa Cruz, Dallas, TX, USA, #sc-74525).