FIGURE 3:

Targeted AMOT depletion reduces phosphorylation of BMP-SMAD proteins specifically. (A–D) MCF7 cells or HUVECs were transfected with siRNA targeting either nonspecific sequences (si-scr) or human AMOT (si-AMOT) and stimulated for the indicated time with 10 nM BMP6 (A–C) or 200 pM TGFβ1 (D). Protein lysates were subjected to Western blotting using the indicated antibodies. Blots represent at least three independent experiments. (B) Quantification depicts pSMAD1/5 level normalized to GAPDH relative to si-scr control. Bar charts represent mean ± SEM from six independent experiments of Western blots depicted in A; **p < 0.01, two-way ANOVA with Bonferroni post-hoc test compared with si-scr stimulated with BMP6 for 30 min. (E) MCF7 cells were transfected with siRNA targeting AMOTL1 (si-AMOTL1) or AMOTL2 (si-AMOTL2) and stimulated with 10 nM BMP6 for 30 min. Lysates were analyzed by Western blotting with the indicated antibodies. The blot is representative for three independent experiments.