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. Author manuscript; available in PMC: 2011 Jul 20.
Published in final edited form as: Dev Cell. 2010 Jul 20;19(1):90–102. doi: 10.1016/j.devcel.2010.06.016

Figure 4. Reduction of Smad1/5/8, but not p38 MAPK, phosphorylation and decreased Runx2 expression in neogenin deficient chondrocytes in response to BMP2.

Figure 4

(A) Decreased BMP2 induced p-Smad1/5/8, but not p-p38 MAPK, in chondrocytes from neogenin mutant mice. Chondrocytes from neogenin+/+ and m/m mice were serum starved for overnight, then stimulated with BMP2 (100 ng/ml) for the indicated time. Cell lysates were analyzed by Western blotting using indicated antibodies. (B-C) Quantitative analysis of data from (A). Phosphorylation of Smad1/5/8 and p38 MAPKwere normalized by total Smad1 and p38 respectively, and quantified by Image J software. Data shown were mean +/− SD, n=3; *, p<0.05, in comparison with control. (D–E) Normal TGF-β and FGF signaling in neogenin deficient chondrocytes. Serum starved chondrocytes were treated with 50 ng/ml TGF-β (D) or 10 ng/ml FGF2 (E) for the indicated time. (F) Reduction of BMP2 induced Runx2 expression in neogenin mutant chondrocytes, which was revealed by real time PCR analysis. Chondrocytes from the wild type and mutant littermates were treated with BMP2 (100 ng/ml) for 2 days. Runx2 transcripts were analyzed by real time PCR and normalized by internal control GAPDH. Data shown were fold over wild type control (mean +/− SD) from 3 independent experiments with duplicate or triplicate samples each; *, p<0.05, significant difference from the wild type control. (G) Rescue of defective BMP reporter expression by neogenin. Wild type and neogenin mutant chondrocytes were transiently transfected vector (control) or neogenin with BMP signaling reporter plasmid (9XSBE-Luc). Transfected cells were stimulated with BMP2 (100 ng/ml). Luciferase activity was normalized and presented as mean +/− S.D. of triplicates from a representative experiment. *, P < 0.05, in comparison with the absence of BMP stimulation. (H) Decreased p-Smad1/5/8 in neogenin mutant growth plates. Cartilage lysates derived from wild type (+/+) and neogenin mutant (m/m) mice at P1 were subjected for Western blot analysis using indicated antibodies. (I) Illustration of a working model for neogenin regulation of BMP signaling and function.