Quantity one 4

The protein concentration of each sample was measured using a bicinchoninic acid (BCA) protein assay kit (Bioteke, China). Equal amounts of total protein (50 μg) from each sample were separated in a 10% SDS-PAGE gel and transferred to a PVDF membrane at 120 V for 2 h at room temperature (RT). The blot was blocked with 5% nonfat dry milk suspended in 1x TBS (25 mM Tris, 137 mM NaCl, and 2.7 mM KCl) for 1 h at room temperature. Membranes were incubated with primary antibodies against STAT6 and GAPDH (Cell Signaling Technology, USA). The resulting blots were incubated with 1:1,000 rabbit–mouse secondary antibodies (Cell Signaling Technology, 4414/4410, USA). Bands were scanned using a densitometer (Bio-Rad) and quantified using the Quantity One 4.6.3 software (Bio-Rad).

MMP-2 activity in culture media samples was assayed using a 0.05% gelatin zymography gel, as described previously (5 (link)). Briefly, 10 µl of each sample was mixed with an equal amount of Laemmli sample buffer (62.5 mM Tris-hydrogen chloride, 25% glycerol, 2% SDS and 0.01% bromophenol blue, pH 6.8) and separated on a 10% SDS-PAGE gel copolymerized with 0.05% gelatin. Enzyme activity was regained by removing the SDS, whereby gels were washed three times for 1.5 h in total in 2.5% Triton X-100 at room temperature following electrophoresis. Washed gels were then bathed in proteolysis buffer (50 mM calcium chloride, 0.5 M sodium chloride and 50 mM Tris, pH 7.8) and incubated at 37°C for 15 h. Following incubation, gels were rinsed in a 2.5% Triton X-100 solution and stained at room temperature with Coomassie blue (45% methanol, 44.75% H₂O, 10% acetic acid and 0.25% Coomassie blue R-250) for 1 h on a rotator. Gels were then destained with a 40% methanol, 7.5% acetic acid and 52.5% H₂O solution until white bands were clearly visible against the Coomassie blue background. Bands were scanned with a densitometer (GS-800; Bio-Rad Laboratories, Inc.) and quantification was performed using Quantity One 4.6.3 software (Bio-Rad Laboratories, Inc.). The experiment was repeated three times, and the relative density values were subjected to statistical analysis.

Whole-cell lysate and the Co-IP supernatant were mixed with 5× SDS-PAGE Plus sample buffer (GenStar, Beijing, China) and then incubated in a boiling water bath before separation by SDS-PAGE and subsequent transfer to PVDF membrane (Millipore, Bedford, MA). The membrane was blocked with 5% skim milk (Solarbio, Beijing, China) for 3 h then incubated with primary antibody overnight at 4°C. The membrane was washed thrice for 10 min each time using Tris-buffered saline and 0.1% Tween-20 (Solarbio, Beijing, China) and incubated with the appropriate secondary antibody. The primary antibodies used for Western blot were β-catenin rabbit polyclonal antibody (1:2,000) (ab6302, Abcam, Cambridge, UK), Myc-tag rabbit monoclonal antibody (1:5,000) (AE070, ABclonal), and HA-tag rabbit monoclonal antibody (1:1,000) (C29F4, Cell Signaling). β-actin rabbit monoclonal antibody (1:2,000) (AF5003, Beyotime) was used as a loading control. The secondary antibody was goat anti-rabbit IgG (1:5,000) (A0208, Beyotime). The blot signal was visualized using ECL reagent (Thermo Fisher Scientific, Carlsbad, CA). The protein expression level was quantified by the band density using Quantity One 4.6.3 software (Bio-Rad Laboratories, Hercules, CA) and normalized by β-actin.

Total RNA (2 µg per reaction) extracted from rat bladder tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA synthesis was performed using the SuperScript III First-Strand kit (Invitrogen) according to the manufacturer's instructions. Synthesized cDNA fragments were amplified by a PCR process in thermal cycler (S1000 Thermal Cycler; BIO-RAD Laboratories, CA, USA).
M2, 3-muscarinic receptors in our study were used published primers (6 (link)15 (link)). PDZ-RhoGEF, LARG, and p115RhoGEF were used published primers (16 (link)). And β-actin primer was designed bound on exon-exon base. The β-actin in the diabetic disease research has been used as a house-keeping gene because the expression of β-actin is not affected by the glucose. Each PCR reaction was carried out with the Maxime PCR PreMix I-StarTaq (iNtRON Biotechnology, Korea) using cDNA equal to 100 ng total RNA, under the following conditions: 94℃ for 2 min and 35 cycles at 94℃ for 30 sec, 60℃ for 30 sec and 72℃ for 30 sec, followed for 7 min. The reaction products were analysed by electrophoresis on agarose gels. Densitometric analysis of band intensity was detected by Gel Documentation System (Gel Doc XR; BIO-RAD Laboratories, CA, USA) and measured using Quantity One 4.6.3 software (BIO-RAD, CA, USA).