Rabbit anti p smad3

The cultured cells were rinsed with cold PBS before treated with RIPA lysis buffer at 4 °C for 10 min. Then the mixture was heated at 100 °C for 10 min and centrifuged under 4 °C at 14000 g min⁻¹ for 10 min. The supernatant was removed, and the protein concentration was measured with the BCA method. About 20 μg of protein was loaded in each lane, separated by 10% SDS–PAGE and transferred to the PVDF membrane. The membrane was blocked with 5% non-fat milk powder for 1 h at room temperature before overnight incubation with primary antibodies 4 °C, followed by the secondary antibody. The antibodies were mouse anti-β-tubulin (Cell Signaling, Danvers, MA, USA; CAT 6181), rabbit anti-MBD3 (Cell Signaling, CAT 3896), mouse anti-Flag (Sigma, San Francisco, CA, USA; CAT F1804), rabbit anti-MMP2 (ImmunoWay, Plano, TX, USA; CAT YT2798), rabbit anti-MMP9 (ImmunoWay CAT YT1892), rabbit anti-Vimentin (Cell Signaling, CAT 5741), rabbit anti-N-cadherin (Cell Signaling, CAT 13116), rabbit anti-E-cadherin (Cell Signaling, CAT 3195), rabbit anti-β-catenin (Cell Signaling, CAT 8480), rabbit anti-Snail (Cell Signaling, CAT 3879), rabbit anti-α-SMA (Cell Signaling, CAT 14968), rabbit anti-Smad2/3 (Cell Signaling, CAT 8685), rabbit anti-P-Smad2 (Cell Signaling, CAT 3108), rabbit anti-P-Smad3 (Cell Signaling, CAT 9520), rabbit anti-TGF-β (Cell Signaling, CAT 3709).

Antibodies were purchased from commercial sources, including rabbit anti-collagen IV from Proteintech (Wuhan, China), mouse anti-glyceraldehyde-3phosphate dehydrogenase (GAPDH) from Novus Biological (Littleton, CO, USA), rabbit anti-fibronectin and rabbit anti-TGF-β1 from Abcam (Cambridge, MA, USA), and rabbit anti-NF-κB-p65, rabbit anti-NF-κB-p-p65, rabbit anti-p-Smad3, and rabbit anti-Smad3 from Cell Signaling Technology (Beverly, MA, USA). Fetal bovine serum (FBS) was procured from Wisent Bioproducts (Saint-Bruno, QC, Canada), D-glucose, D-mannitol and Streptozotocin (STZ) from Sigma-Aldrich (Saint Louis, MO, USA), and Trizol from Life Technologies (Carlsbad, CA, USA), dulbecco’s modified eagle medium (DMEM) from Thermo Fisher Scientific (Waltham, MA, USA). Albumin ELISA kit was purchased from Abcam (Cambridge, USA), and creatinine assay kit was from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Wogonin was obtained from Topscience (Shanghai, China); Horseradish-peroxidase (HRP)-conjugated goat anti-rabbit/mouse IgG and 3.3-diaminobenzidine (DAB) was from Beijing Zhongshan Biotechnology (Beijing, China).