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Fig. 6. Id1 inhibits Twist-1 function by sequestering E47 from Twist-1. (A) COS-7 cells were transiently transfected with Flag-Twist-1, Myc-tagged E47 and increasing doses (0.5, 1 or 2 µg) of Myc-tagged Id1 construct. Lysates were immunoprecipitated using anti-Flag antibody and blotted with anti-Flag and anti-Myc-antibody. (B) COS-7 cells were transiently transfected with Flag-Id1, Myc-tagged E47 and increasing doses (0.5, 1 or 2 µg) of Myc-tagged Twist-1 construct. Lysates were immunoprecipitated using anti-Flag antibody and blotted with anti-Flag and anti-Myc-antibody. (C) Endogenous E47 in C3H10T1/2 cells was precipitated using anti-E47 antibody. Then, Id1 was detected in the immunoprecipitates by western blot. (D) P19 cells were transiently transfected with 3GC2-Lux luciferase construct in combination with 50 ng of BMPR-IB(QD), Smad1 and Smad4, 25 ng of Twist-1 and E47, and increasing doses (25 or 100 ng) of Id1 expression construct. Cells were lysed and luciferase activity was assayed 24 hours after transfection (*P<0.01). (E) MC3T3-E1-Tw1 cells were transiently transfected with either Id1 or GFP expression construct by electroporation (Amaxa). The cells were grown in the presence or absence of rhBMP2 (300 ng/ml) for 6 days. ALP activity was measured as described in the Materials and Methods. Data are presented as mean ± s.d. of triplicate samples (*P<0.05).
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