Anti vimentin antibody

Total proteins were extracted and separated using SDS‐PAGE gels. Anti‐SETD2 antibody (Invitrogen), anti‐E‐cadherin antibody (Cell Signaling Technology), anti‐vimentin antibody (Abcam), and H3K36me3 antibody (Cell Signaling Technology) were used at a concentration of 1:2000. H3 antibody (Cell Signaling Technology, 1:2000) and GAPDH antibody (Santa Cruz Biotechnology, 1:2000) were used as loading controls.

Proteins were obtained from HK-2 cell lysates and animal tissue, using RIPA buffer (Thermo, USA) containing Complete, Mini Protease Inhibitor Cocktail (Roche, USA) and Halt Phosphatase Inhibitor Cocktail (Thermo, USA). To examine the expression of proteins, separated protein in SDS/PAGE (gradient gels) were transferred onto polyvinylidene difluoride membranes (PVDF) (Bio-rad, USA), and then incubated with 5% BSA (sigma) for blocking. The membranes were incubated with anti-GAPDH antibody (Santacruz, USA), anti-type 1 collagen antibody (abcam, UK), anti-fibronectin antibody (abcam), anti-α-SMA antibody, anti-Vimentin antibody, anti-Lin28a antibody, anti-phospho-ERK antibody, anti-ERK antibody, anti-phospho-JNK antibody, anti-JNK antibody, anti-phospho-p38 antibody, anti-p38 antibody, anti-phospho-smad3 antibody and anti-smad3 antibody (Cell signaling, USA). After incubating with HRP-linked antibody (Cell signaling), the protein expression on blots was detected by ChemiDoc^TMXRS+ (Bio-rad) and the bands were quantified using the Image Lab^TM Software (Bio-rad).

Total protein samples were extracted form cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) with PMSF. Protein concentration was quantified using a BCA kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. The Membranes were then blocked with 5% non-fat milk for 1 h at room temperature, and incubated with the primary antibodies overnight at 4°C. Then, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies for 1 h at room temperature. The protein bands were visualized using an ECL kit (Beyotime Institute of Biotechnology). The gray values of the protein bands were quantified using ImageJ 1.51 software (National Institutes of Health) and relative expression levels were calculated. The following antibodies were employed: Anti-NASP (1:1,000; cat. no. ab181169; Abcam), anti-N cadherin (1:3,000; cat. no. ab76011; Abcam), anti-E cadherin (1:1,000; cat. no. ab231303; Abcam), anti-vimentin antibody (1:1,000; cat. no. ab16700; Abcam), anti-GAPDH (1:5,000; cat. no. ab8245; Abcam), goat anti-rabbit (1:3,000; cat. no. 7074; CST Biological Reagents Co., Ltd.) and horse anti-mouse (1:3,000; cat. no. 7076; CST Biological Reagents Co., Ltd.).