Filamin a

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).