FIGURE 4.

GADD45β knockdown decreases p38 phosphorylation and Col10a1 mRNA levels, without affecting C/EBPβ binding to the Col10a1 promoter in vivo. A, siGFP and si45β-ATDC5 cells were differentiated for 1 week (1w) and 2 weeks (2w). Chromatin was cross-linked and enzymatically sheared, and after reverse cross-linking of the DNA-protein complexes, the precleared lysates were incubated with antibodies against C/EBPβ (+) or normal rabbit IgG (−) overnight at 4 °C. The mouse Col10a1 promoter region was PCR-amplified using primers spanning from −114 to +91, and the PCR products were resolved on a 2.5% agarose gel. Data are representative of two independent experiments performed in duplicate. B, total RNA isolated from 2-week differentiated siGFP- and si45β-ATDC5 was analyzed by real-time PCR. Each value was normalized to β-actin in the same sample and shown as mean ± S.D. *, p < 0.05. C, the siGFP- and si45β-ATDC5 cells were incubated with 100 ng/ml BMP-2 for the indicated times, and total lysates were analyzed on Western blots using antibodies against phospho-p38 (P-p38), total p38 (p38), and β-actin.