α smooth muscle actin α sma

Kidney tissue and NRK-52E cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) containing PMSF and centrifuged at 12,000 × g for 20 min at 4 °C. Equal amounts of protein (renal tissue protein: 20 μg, cellular proteins: 15 μg) were separated by SDS-PAGE, the gel was transferred to a PVDF membrane, and blocked with rapid blocking solution (Xin Saimei) for 10 min. The membranes were incubated at 4 °C with primary antibodies against NLRP3 (1:1000, Proteintech, Rosemont, IL), caspase-1 (1:500, Proteintech, Rosemont, IL), GSDMD (1:1000, GeneTex, Irvine, CA), IL-1β (1:500, Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (1:1000, Proteintech, Rosemont, IL), vimentin (1:1000, Proteintech, Rosemont, IL), and fibronectin (1:1000, Proteintech, Rosemont, IL) overnight. The membranes were washed three times with TBST for 10 min, diluted secondary antibody (1:10,000, Zenbio, Durham, NC) was added, and the membranes were incubated at room temperature for 1 h. ECL developer (Sorfa, Zhejiang, China) was evenly dropped on PVDF membranes, developed and saved. The gray level of the spots was analyzed with Image Lab software. β-actin was used as an internal reference.

Radioimmunoprecipitation assay buffer was used for preparing whole cell lysates. Protein was separated by sodium dodecyl‐sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore). In addition, 1% bovine serum albumin (BSA) was added to block the membranes. Then, the membranes were washed three times with TBS‐0.1% Tween 20 (TBST). The washed membranes were incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: anti‐GAPDH (1:30,000, Cat: 60004‐1‐Ig; Proteintech), Collagen‐I (1:1000, Cat: 14695‐1‐AP; Immunoway), Collagen‐III (1:500, Cat: 22734‐1‐AP; Proteintech), α‐smooth muscle actin (α‐SMA) (1:3000, Cat: 14395‐1‐AP; Proteintech), anti‐Actin (1:5000, Cat: BS6007M; Bioworld), anti‐CXCL4 (1:3000; Proteintech), anti‐TGF‐β1 (1:1000, Cat: CPA2154; Cohesion), and anti‐p‐Smad2/3 (1:1000, Cat: BS1838; Bioworld). The membranes were then incubated with the following horseradish peroxidase‐conjugated secondary antibodies: Afterwards, the secondary antibody (1:10,000; Proteintech) was incubated at room temperature for 1 h. Finally, the antigen‐antibody reactions were visualized by chemiluminescence (ECL) kit, and the intensity of protein bands was quantified by using ImageJ software.

The left ventricle under the ligation was used to measure the expression of protein. The whole experimental process of the Western blotting was performed as previously described (Verma et al., 2021 (link)). Antibodies used were as following: Tublin (cat. no. 00082687, Proteintech), α-Smooth muscle actin (α-SMA) (cat. no. 14395-I-AP, Proteintech), Collagen Ⅲ (cat.no. 30565S, Cell Signaling Technology), and Collagen Ⅰ (cat.no. NB600-450, Novus). The result was obtained using an ECL detection kit (Millipore, United States).

Kidney tissue or cells were lysed in lysis buffer containing 2% SDS following centrifugation. The supernatant was separated on SDS‐polyacrylamide gels. Then, proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membrane (Millipore) and blocked with 5% non‐fat milk. Proteins were detected with primary antibody against E‐cadherin (610181; BD Transduction Laboratories), Snail (3879; Cell Signaling Technology), Fibronectin (F3648; Sigma‐Aldrich), FoxM1 (ab1807.10; Abcam), collagen I (Col I) (ab34710; Abcam), Wnt1 (ab15251; Abcam), Wnt2b (ab15251; Abcam), Wnt3 (ab172612; Abcam) from Abcam (Cambridge, MA, USA), active β‐catenin (05‐665; Millipore), β‐catenin (51067‐2‐AP; Proteintech), Col I (14695‐1‐AP; Proteintech), α‐smooth muscle actin (α‐SMA) (14395‐1‐lg; Proteintech), GAPDH (60004‐1‐lg, Proteintech) from Proteintech. The bands were visualized by an ECL detection kit (AmerSham Biosciences) and quantitated via densitometric analysis using the Image J software (NIH).