Nedd4l

Cycloheximide was purchased from Beyotime Biotechnology (Hangzhou, China). MG132 was purchased from Sigma-Aldrich (USA). Rhodamine phalloidin was purchased from Cytoskeleton (USA). A G-LISA RhoA Activation Assay Biochem Kit was purchased from Cytoskeleton. Antibodies against AAMP (used for western blot [WB]) and RhoA (used for WB and IHC staining) were purchased from Acbam. Antibodies against β-actin, p-ERM, N-cadherin, E-cadherin, Snail, Ubiquitin, NEDD4, NEDD4L, and SMURF2 were obtained from Cell Signal Technology. Antibody against AAMP (used for IHC staining) was obtained from GeneTex. Antibody against RhoA (used for IP) was purchased from Santa Cruz Biotechnology.

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).

Antibodies for phospho‐ERK1/2 (#4370S), phospho‐JNK (#9251S), phospho‐p38 (#4511S), phospho‐p65 (NF‐κB) (#3033 s), ERK1/2 (#4696), JNK (#9251 s), p38 (#8690), NF‐κB p65 (#8242), and NEDD4L (#4013) were from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA). Antibodies for TRAF2 (#5525–1), MEKK3 (#1673–1), and MEKK5 (#1772–1) were from Epitomics (Epitomics, Burlingame, CA). Antibodies for TAK1 (SC‐7967), MEKK2 (SC‐1088), TRAF3 (SC‐949), and TRAF6 (SC‐7221) were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA). Phospho‐MEKK2 Ser520‐specific antibody was prepared in Abmart (Shanghai, China). Human or mouse IL‐17A/F (human IL‐17, #200–17; Murine IL‐17A, #210–17; human IL‐17F, #200–25; and murine IL‐17F, #210‐17F) were purchased from PeproTech (PeproTech, Rocky Hill, NJ). E1, UBCH5b, and ubiquitin recombinant proteins were gifts from Prof. Zongping Xia (Zhejiang University).