FIGURE 4:

AMOT depletion causes decreased BMP-SMAD signaling. (A–D) MCF7 cells were transfected with siRNA targeting either nonspecific sequences (si-scr) or human AMOT (si-AMOT) and stimulated for the indicated time points with 10 nM BMP6. (A) Endogenous complex formation of SMAD1 and SMAD4. After treatment, MCF7 cells were lysed and immunoprecipitation using αSMAD1 antibody was performed. Precipitates and TCL were analyzed by Western blotting using the indicated antibodies. Rabbit IgG served as control. (B) After 1 h of stimulation, MCF7 cells were lysed and their cytosolic and nuclear proteins fractionated and blotted with the indicated antibodies. The blot represents three independent experiments. (C) After 1 h of stimulation, cells were lysed and RNA was extracted, reverse transcribed, and used for gene expression analysis. qRT-PCR analysis of ID1 mRNA. Data are presented as mean ± SEM of three independent experiments; ***p < 0.001, two-way ANOVA with Bonferroni post-hoc test, compared with si-scr stimulated with BMP6 for 1 h. (D) Representative Western blot of MCF7 protein lysates after AMOT depletion and 1 h BMP6 stimulation shows ID1 and GAPDH level.