Trpc6

Protein extraction from mouse kidney tissues and HK‐2 cells was performed with radio immunoprecipitation assay (RIPA) lysis buffer. Nucleoprotein and plasma protein extraction was performed using the nuclear and cytoplasmic protein extraction kits (Beyotime Institute of Biotechnology, China) according to the manufacturer's instructions. The protein concentration was determined by using a BCA protein assay kit (Thermo Fisher Scientific). Proteins were separated using 10% SDS‐PAGE and transferred to PVDF membranes, which were probed with primary antibodies against NFATc1 (1:1000 dilution, Abcam), TRPC6 (1:1000 dilution, Abcam), fibronectin (1:1000 dilution, Abcam), collagen 1 (1:1000, Affinity), cleaved caspase 3 (1:1000, CST) and IL‐6 (1:1000 dilution, Proteintech). β‐actin (1:5000, Proteintech) and lamin B1 (1:5000, Proteintech) were used as internal controls. Antibody‐antigen complexes were detected with enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech, Uppsala, Sweden) after incubation with a suitable secondary antibody as described previously.²⁷, ²⁸

Protein detection was performed in RIPA buffer (Sigma) lysed VIC. Total protein levels were quantified with a Pierce BCA protein assay (Thermo Scientific, Hemel Hempstead, UK). Protein lysates (7.5 μg) were electrophoretically separated under denaturing conditions on 10% Bis-Tris gels (Invitrogen, Renfrew, UK), and transferred on to nitrocellulose membranes (Hybond C, Amersham, UK). Specific MSC were detected using following antibodies: Kir6.1 (Bioss, London, UK), TREK-1, TRPM4, TRPV4, TRPC6 (Abcam, Cambridge, UK), followed by washing and incubation with secondary antibodies. Bands were visualized using enhanced chemiluminescence substrate and positivity was captured on Hyperfilm (GE Healthcare, Amersham, UK). Films were scanned and bands were quantified using the QuantityOne program (Biorad, Hercules, USA). Levels of protein expression were normalised to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (R&D Systems, Abingdon, UK).

Proteins were separated on 7.5% SDS-polyacrylamide gels and blotted onto PVDF membranes. Membranes were blocked with 5% milk and incubated with the following primary antibodies: Ca_V1.2 (Neuromab), PKD1 and PKD2 (Santa Cruz, sc-100415), ANO1, TRPC6 and TRPM4 (Abcam) and actin (MilliporeSigma) overnight at 4°C. Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature. Protein bands were imaged using an Amersham Imager 600 gel imaging system (GE Healthcare) and quantified using ImageJ software.

Eight-week-old male ApoE^−/− mice were housed under standard animal room conditions (temperature, 21 ± 1°C; humidity, 55–60%). The animals were randomly divided into two groups: the normal-diet group and the HFD group. The animals of the HFD group were subsequently maintained on diet with high fat for 12 weeks to induce atherosclerosis. After twelve weeks, the aortas were carefully excised from the mice. The aortic roots along with the basal portion of the heart were fixed with 4% paraformaldehyde, followed by embedding in OCT compound, and were cut cross-sectionally into 7-μm-thick sections. Atherosclerotic lesions of the aortic root were observed by HE staining. Oil red O staining was performed according to the manufacturer's instructions to show the lipid deposition with an Oil red O staining kit (Nanjing Jiancheng Biology Engineering Institute, Nanjing, Jiangsu, China). In addition, immunostaining was performed on the aortic root for expression of TRPC6 (Abcam, Cambridge, MA, USA). VE-cadherin (Abcam, Cambridge, MA, USA) was used as an endothelial marker. Images were captured using a confocal laser scanning microscope. Fluorescence intensity of TRPC6 was measured using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA). The data were calculated from 5 mice for each group. For each mouse, 5 cells were randomly selected for quantification.