Figure 5.

Samd4 (sterile alpha motif domain containing protein 4) bound to mitogen-inducible gene 6 (Mig6) mRNA. (a) Western blotting analysis of RNA immunoprecipitation (IP) product reveals Samd4 was enriched in Flag IP. (b) Top, DNA structure of 5′ untranslated region (UTR) of Mig6. Bottom, density plot showing the distribution of Flag IP and immunoglobulin G (IgG) peaks according to this region. (c) The protein levels of SAMD4 and MIG6 in primary osteoblastswere assessed by western blotting after cells were infected with GFP- or Samd4-expressing lentivirus. (d, e) The protein (d) and mRNA level (e) of Mig6 in calvarium from 6-day-old wild-type (WT) and Samd4^PB/PB mice were assessed by western blotting and quantitative PCR, respectively. (f) Immunohistochemistry analysis of MIG6 expression on proximal tibia from 1-week-old Samd4^PB/PB mice and control WT mice. (g) The representative photographs of fast blue staining and alizarin red staining of osteoblasts infected by GFP- or MIG6-expressing lentivirus. (h) The representative photographs of fast blue staining of osteoblasts differentiated from WT and Samd4^PB/PB osteoblasts infected with shRNA lentivirus targeting GFP or Mig6, cultured in OBD for 7 days. (i) Quantitative parameters of Alp activity analyzed by colorimetric assay. Values represent mean±s.d. (n=3 in panel (e) and 6 in panel (i) for each genotype). P-values were obtained from t-tests with paired or unpaired samples, *P<0.05, **P<0.01. Bars=100 μm in panel (f) and 2 mm in panels (g and h).