Fig. 6

COL2A1 phosphorylated SMAD1^S206 by activating ERK1/2, which negatively influenced BMP-SMAD1 activity. a, b Immunoblotting evaluation of p-SMAD1^S463/465, p-SMAD1^S206, and SMAD1 in chondrocytes from Col2a1 mutant mice (a) and SW1353 and Hs819.T cells treated with 100 μg·mL⁻¹ COL2A1 or vehicle (0.05 mol·L⁻¹ acetic acid) for 1 h (b). c, d Immunoblotting evaluation of p-ERK1/2, ERK1/2, p-MAPK14, MAPK14, p-JNK1/2, and JNK1/2 in chondrocytes from Col2a1 mutant mice (c), and SW1353 and Hs819.T cells treated with COL2A1 or vehicle (d). e−g The expression levels of p-ERK1/2, p-SMAD1^S463/465, p-SMAD1^S206, ERK1/2, and SMAD1 proteins were detected by immunoblotting (e), and the expression levels of ID1, ID2, DLX5, RUNX2, and COL10A1 were tested by qPCR in SW1353 (f) and Hs819.T cells (g) pretreated with 10 μmol·L⁻¹ U0126 or vehicle 1 (dimethylsulfoxide) for 1 h and then treated with 100 μg·mL⁻¹ COL2A1 or vehicle 2 (0.05 mol·L⁻¹ acetic acid) for 1 h. h, i Immunoprecipitation was performed on 293T cells treated with COL2A1 or vehicle with anti-SMAD1 antibody (h) or anti-ERK1/2 antibody (i), followed by immunoblotting with anti-ERK1/2 and anti-SMAD1 antibodies. j Immunoblotting evaluation of SMAD1 in both nuclear (Nuc) and cytoplasmic (Cyto) extracts in SW1353 and Hs819.T cells pretreated with U0126 or vehicle 1 and then treated with COL2A1 or vehicle 2. k, l Immunoprecipitation was performed on 293T cells treated with COL2A1 or vehicle with anti-SMAD1 antibody (k) or anti-SMAD4 antibody (l), followed by immunoblotting with anti-SMAD4 antibody and anti-SMAD1 antibody. Data in (f) and (g) are presented as the mean ± SD (n = 3). *P < 0.05