Figure 2. Cloning and characterization of Fam57b2 from Cyp24a1-null callus tissue.

(A) Binding of [³H]- 24R,25(OH)₂D₃ to cDNAs identified by microarray analyses. **P < 0.01, by 1-way ANOVA followed by Dunnett’s post test (n = 3). (B) Saturation binding analysis (1-site–specific binding with Hill slope; n = 3). (C and D) Binding competition analysis. **P < 0.01 and ***P < 0.001, by 1-way ANOVA followed by Dunnett’s post test; 1-site fit K_i (n = 3). Pg, progesterone. (E and F) Fam57b isoform expression in tissues. Relative expression levels were determined by reverse transcription quantitative PCR (RT-qPCR) and normalized to B2m. Expression in brain (Br) was arbitrarily assigned a value of 1. LB, long bones; SI, small intestine; Cal, calvaria; Cart, cartilage; Hrt, heart; Kid, kidney; Mus, skeletal muscle. Relative expression is indicated by numerals above each bar. (G and H) Fam57b isoform expression in fracture callus. Relative expression levels were determined at intervals by RT-qPCR and normalized to B2m; expression in contralateral tibia (No OT) was arbitrarily assigned a value of 1. *P < 0.05, by 2-way ANOVA followed by Bonferroni’s post test. (I–L) Expression of Fam57b isoforms following 24R,25(OH)₂D₃ treatment in callus (I and K) or primary cultures of chondrocytes (J and L). *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way ANOVA followed by Bonferroni’s post test (n = 6). The number of animals per group is indicated in parentheses.