β actin

The bladder tissues were homogenized using tissue grinders (Shanghai Jingxin, Shanghai, China) at 65 Hz for 2 min to extract the total protein. BCA protein assay Kit (Beyotime Biotechnology, China) was used to measure the protein concentration. Equivalent proteins (20 μg) were subjected to 10% or 8% SDS-PAGE at 80 V for 30 min or 120 V for 60 min, respectively, to separate the proteins of different molecular weights and transfer to the PVDF membranes using the transblotting apparatus (Bio-Rad Laboratories, Hercules, CA, United States) for 55 or 110 min, respectively, at 300 mA. The PVDF membranes were blocked with 5% (w/v) non-fat milk buffer at room temperature for 2 h and incubated with a primary antibody in TBST [Myosin Va (1:1000, Santa Cruz), SLC17A9 (1:1000, MBL), or β-actin (1:1000, 4A Biotech)] overnight at 4°C. The immune-labeled membranes were washed three times with TBST for 15 min each time, and then conjugated with a secondary antibody (1:5000, 4A Biotech) at room temperature for 2 h. After the non-binding secondary antibodies were washed away, the target protein bands were visualized using a chemiluminescent reagent (Millipore, United States). Data were processed using ImageJ, and the immunoblot protein expression levels of myosin Va and SLC17A9 were normalized using β-actin. The antibodies used in the present study are listed in Supplementary Table 1.

Total proteins were extracted from liver, adipose tissue, and skeletal muscle with Radio-Immunoprecipitation Assay (RIPA) lysis buffer (Bioteke, Beijing, China) and separated by sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were subsequently transferred to polyvinylidene difluoride membranes (Millipore, Billerica, USA). The blots were blocked with 5% nonfat milk solution for 1.5 h at room temperature and then incubated over night at 4°C with the antibody against ACE2 (Epitomics, California, USA) and incubated at room temperature with peroxidase conjugated goat anti-rabbit IgG secondary antibody (MultiSciences Biotech Co., Ltd., Hangzhou, China). Immunoreactivity was detected by enhanced chemiluminescence detection kit (Keygen, Nanjing, China). The band density of ACE2 was normalized to the corresponding density of β-actin (4A Biotech Co., Ltd., Beijing, China).