Vimentin

Renal tissues or cells were harvested and centrifuged at 12,000 g to remove cell debris. Protein concentration was measured using a BCA assay kit (Pierce). Eighty micrograms of protein was subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After transfer, the membranes were blocked and blotted routinely with antibodies against VE-cadherin (BD Biosciences), α-SMA (Abcam), collagen1 (Abcam), PP2Ac (Cell Signaling Technology), occludin (Invitrogen), P-threonine/P-serine (Santa Cruz), nitrotyrosine (Cayman), acetylated-lysine (Cell Signaling Technology), phosphotyrosine (PY-20,Biolgend), PP2A,C subunit, demethylated (Millipore), vimentin (Boster) and CD31 (Proteintech). The immunolabeled proteins were detected by enhanced chemiluminescence (Pierce).The density of the bands was analyzed by Quantity One software (Bio-Rad).

Experiments were performed according to protocols previously described [31 (link)]. Samples were incubated overnight at 4⁰C with different dilutions for immunofluorescence with primary antibodies against ClC-3 (1:50, Abcam), β1 integrin (1:100, BD Pharmingen), α-tubulin (1:50, Abcam), vimentin (1:100, Boster), K18 (1:100, Boster) and K18 pS52 (1:50, Abcam). All antibodies were diluted to 1:1000 for immunoblotting.

Total tissues and cellular proteins were extracted and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes incubated overnight at 4 °C with primary antibodies. The antibodies against CDK2, Cyclin D1, β-catenin, C-myc, N-cadherin, Snail were purchased from Affinity (USA), p21 antibody was from Cell Signaling Technology (USA), E-cadherin antibody was from BD Biosciences (USA), antibodies against MMP9, Vimentin, β-actin and GAPDH were from Boster (China).

Protein was extracted and separated by SDS-polyacrylamide gel electrophoresis and was then transferred to polyvinylidene ethylene membranes, blocked from the nonspecific antibody binding sites with non-fat milk powder (concentration of 0.5%) at room temperature for 2 h, and then polyclonal antibodies against α-SMA (1 : 200, Boster, China), vimentin (1 : 200, Boster, China), TGF-β1 (1 : 200, BioVision, USA), TGF-β3 (1 : 100, Abcam, USA), and GAPDH (1 : 5000, Boster, China) were added. This was followed by incubation overnight at 4°C, followed by incubation with a secondary antibody (1 : 5000) for 2 h, and then autoradiograms were taken with ECL reagent in the chamber. Finally, imaging analysis was carried out by an automated electrophoresis gel imaging system.

The apexes of the hearts were isolated from 1 ~ 3-day-old Sprague-Dawley rats which were obtained from the Experimental Animal Center of Bengbu Medical College (Bengbu, China). All the animal procedures were in accordance with the United States National Institutes of Health Guide and were approved by the Animal Use and Care Committee of Bengbu Medical College.
After washing in precooling D-Hank's solution, the heart tissues were sheared and fully digested (37°C, 5% CO₂, incubated for 7 ~ 8 min). The digestive enzymes consisted of trypsin (0.07%, Beyotime Biotechnology, Shanghai, China), type II collagenase (0.08%, Sigma-Aldrich Co., St. Louis, MO, USA), and DNase I (10 μg/mL, Beijing Solarbio Science & Technology Co. Ltd., Beijing, China). When the tissue started to loosen, precooling DMEM medium (10% FBS) was added to stop the digestion. Cells isolated from the tissue were collected and cultured in DMEM medium (glucose 5.5 mM) containing 10% FBS in an incubator (37°C, 5% CO₂) for 90 min. Vimentin (1 : 200, Boster Biological Technology, Wuhan, China)-positive cells were considered as CFs. As the cells grew to 80% confluence, they were passaged at a ratio of 1 : 2, and the 2nd to 4th passages of cells were used for the following experiments.