Figure 6.

TNFα accelerates NOTCH2 signal activation during osteoclast differentiation and enhances JAG1 expression. BMMs derived from 2-month-old Notch2^tm1.1Ecan mice and control littermates were cultured in the presence of M-CSF at 30 ng/ml and of TNFα at 200 ng/ml for 6 days. A, total RNA was extracted, and gene expression was determined by qRT-PCR. Data are expressed as Jag1, corrected for Rpl38 copy number. Values are means ± S.D.; n = 4 biological replicates for control (white bars) and Notch2^tm1.1Ecan (black bars) cells. *, significantly different compared with day 0, p < 0.05. B, whole-cell lysates (35 μg of total protein) were examined by immunoblotting using anti-JAG1, NOTCH2 and N2ICD, HES1, NFATc1, and β-Actin antibodies. The band intensity was quantified by ImageLab^TM software (version 5.2.1), and the numerical ratio of JAG1/β-Actin, NOTCH2/β-Actin, N2ICD (including N2ICD^ΔPEST)/β-Actin, HES1/β-Actin, and NFATc1/β-Actin is shown below each blot. All control ratios at day 0 are normalized to 1.