Fig. 4

The ITGB1 receptor mediated the effect of COL2A1 on the BMP pathway and chondrocyte hypertrophy. a, b Immunoprecipitation with anti-BMPR1A (a) or anti-BMPR1B (b) antibody was performed in extracts of 293T cells treated with 100 μg·mL⁻¹ COL2A1 or vehicle (0.05 mol·L⁻¹ acetic acid) for 1 h, and then, immunoblotting was conducted with anti-phospho-serine/threonine and anti-BMPR1A/B antibodies. c−e Immunoblotting evaluation of p-ITGB1 and ITGB1 in chondrocytes of Col2a1 p.Gly1170Ser mutant mice (c), SW1353 and Hs819.T cells treated with COL2A1 or vehicle (d), and human articular chondrocytes transfected with COL2A1 siRNAs or negative control siRNA (e). f−h SW1353 and Hs819.T cells were pretreated with 10 μg·mL⁻¹ ITGB1 blocking antibody or anti-human IgG antibody for 1 h and then treated with COL2A1 or vehicle for 1 h. Immunoblotting evaluation of p-ITGB1, p-SMAD1^S463/465, ITGB1, and SMAD1 (f), and qPCR evaluation of ID1, ID2, DLX5, RUNX2, and COL10A1 (g, h) were performed. i Immunoblotting evaluation of SMAD1 in both nuclear (Nuc) and cytoplasmic (Cyto) extracts was performed in SW1353 and Hs819.T cells pretreated with ITGB1 blocking antibody or anti-human IgG and then treated with COL2A1 or vehicle. Data in (g) and (h) are presented as the mean ± SD (n = 3). *P < 0.05. AC, articular chondrocyte; Blk ab, blocking antibody