Figure 5. Stabilization of 3BP2 is required for activated osteoclasts and TNF-α production in Rnf146^fl/fl LysM-Cre osteoclast progenitors.

(A) Whole cell lysates from primary murine osteoclast progenitors derived from WT and Rnf146^fl/fl LysM-Cre (KO) mice were probed with the indicated antibodies for Western blot analysis. (B and C) TRAP staining (B) or resorption pit assay (C) of osteoclasts derived from WT, Rnf146^fl/fl LysM-Cre (KO), and Rnf146^fl/fl Sh3bp2^fl/fl LysM-Cre (dKO) osteoclast progenitors cultured in the presence or absence of RANKL for 7 days. Original magnification, ×40. n = 3. (D) Whole cell lysates from cells in B were probed with the indicated antibodies for Western blot analysis. (E–G) qPCR analysis of Ctsk (E), Calcr (F), and Acp5 (G) mRNA expression in primary murine osteoclast progenitors derived from WT, Rnf146^fl/fl LysM-Cre (KO), and Rnf146^fl/fl Sh3bp2^fl/fl LysM-Cre (dKO) mice and cultured in the presence of RANKL for 0 to 3 days. The relative expression of each mRNA was normalized to that in WT cells at day 3. n = 3. (H) qPCR analysis of Tnfα mRNA expression in primary murine osteoclast progenitors derived from WT, Rnf146^fl/fl LysM-Cre (KO), and Rnf146^fl/fl Sh3bp2^fl/fl LysM-Cre (dKO) mice and cultured in the presence of LPS (10 ng/ml) for 0 to 24 hours. n = 3. (I) WT, Rnf146^fl/fl LysM-Cre (KO), and Rnf146^fl/fl Sh3bp2^fl/fl LysM-Cre (dKO) mice were injected i.p. with LPS, and TNF-α in the serum was measured by ELISA 4 hours later. n = 6. P values were determined by ANOVA with Tukey-Kramer’s post hoc test (B–I) or unpaired t test (A). Data are presented as mean ± SEM. *P < 0.05.