Rabbit anti smad2 3

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).