Image quant las 4000 bioimaging system

Rat bladders were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) to extract total protein. Protein concentrations were measured with a Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, USA). Then, 50 μg protein aliquots were separated using 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). After being blocked with 5% skim milk dissolved in Tris-buffered saline at room temperature for 2 h, the membranes were incubated overnight at 4 °C with various primary antibodies targeting the following proteins: IL-6 (Abcam, Cambridge, UK, ab9324, 1:800), TNF-α (Abcam, ab199013, 1:500), Piezo1 (Alomone labs, Jerusalem, Israel, APC-087, 1:500), NCX1 (Abcam, ab2869, 1:800), NCX2 (Alomone labs, ANX-012, 1:500), NCX3 (Alomone labs, ANX-013, 1:500), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, AG019, 1:1000), and α-tubulin (Beyotime, AT819, 1:1000). Following incubation with horseradish peroxidase-conjugated secondary antibodies (Zhongshan Co., Beijing, China, ZB-2301, ZB-2305, 1:5000), antibody–antigen complexes were detected using an ECL substrate (Millipore, Billerica, USA) and visualized with an Image Quant LAS-4000 BioImaging System (GE Healthcare, Stockholm, Sweden).

For western blotting, total protein was extracted from mouse bladders and clinical specimens using RIPA lysis buffer (Beyotime, Shanghai, China), and protein concentrations were measured using a Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Fifty micrograms of protein was then loaded onto SDS-PAGE gels and separated before being transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). After being blocked with 5% bovine serum albumin dissolved in Tris-buffered saline for 2 h, the membranes were incubated with primary antibodies to the following proteins overnight at 4 °C: IL-6 (GeneTex, San Antonio, TX, USA,GTX110527, 1:1000), TNF-α (GeneTex, GTX110520, 1:1000), HCN1 (Abcam, Cambridge, UK, ab84816, 1:1000), HCN2 (Abcam, ab65704, 1:1000), HCN3 (Abcam, ab84818, 1:1000), HCN4 (Abcam, ab69054, 1:1000), Filamin A (Cell Signaling Technology, Beverly, MA, USA, 4762, 1:1000), NEDD4L (Cell Signaling Technology, 4013, 1:1000), α-tubulin (Beyotime, AT819, 1:1000) and GAPDH (Beyotime, AG019, 1:1000). Horseradish peroxidase-conjugated species-specific secondary antibodies (Zhongshan Co., Beijing, China, ZB-2301, ZB-2305, 1:5000) were used to detect the above primary antibodies. The proteins were visualized using ECL Substrate (Millipore, Billerica, MA, USA) and detected using an Image Quant LAS-4000 BioImaging System (GE Healthcare, Stockholm, Sweden).