Transblotting apparatus

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.

Pretreated bacteria or purified protein were solubilized and loaded onto SDS polyacrylamide gels. After electrophoresis, samples were stained with Coomassie brilliant blue, or transferred (2 h at 100 V) to a polyvinylidene difluoride membrane (Roche Diagnostics, German) using a transblotting apparatus (Bio-Rad, USA). The membrane was blocked in 5 % skimmed milk-TBST at 4℃ overnight. The membrane was incubated with His-tag (4C2) monoclonal antibody (1:8,000; Bioworld Technology, China) at RT for 1 h followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:20,000; Proteintech, China) at RT for 1 h, and visualized with enhanced chemiluminescence (CWBio, China).

The tissue was homogenized, and total proteins were extracted using a total protein extraction reagent kit [24 ]. The protein concentration was measured by using a BCA protein assay kit (Pierce, USA). Protein samples were separated on SDS-PAGE gels at 70–90 V and transferred to PVDF membranes by using a transblotting apparatus (Bio-Rad Laboratories, USA) for 70 min at 90 V. The membranes were blocked with 5 % (w/v) non-fat milk at room temperature for 90 min and subsequently incubated overnight at 4 °C with rabbit anti-TRPV1 (1:1000; Abcam). The immune-labeled membranes were washed once with PBST for 15 min, followed by two separate washes (5 min/wash). The membranes were then probed with secondary antibody (1:2000; Millipore) at room temperature for 60 min in 5 % non-fat milk. The membrane was washed three times with PBST, and protein bands were visualized with ECL Western blotting detection reagents (Bio-Rad Laboratories, USA). The intensity of each target protein band was analyzed using an Image Station 4000R (KODAK, USA) and expressed relative to β-actin density.

The purified fusion protein was solubilized in sample buffer (50 mM Tris·HCl pH 6.8, 10% glycerol, 0.1% bromophenol blue, 1% 2-mercaptoethanol, 2% SDS) and loaded onto SDS polyacrylamide gels. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membrane (Roche Diagnostics) for 2 h at 100 V using a transblotting apparatus (Bio-Rad, USA). The membrane was blocked overnight at 4 °C in 5% skimmed milk-TBST and was detected by GST-Tag monoclonal antibody (3A10) (Bioworld Technology, China) with a 1:8000 dilution at RT for 1 h. The primary antibody binding was incubated with a 1:20000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (Proteintech, China) at RT for 1 h and visualized with an enhanced chemiluminescence kit (CWBio, China).