Figure 4. Endogenous Ring1B is required to sustain steady state levels of Fak, modulate Erk phosphorylation and to allow Fak recruitment to focal adhesions upon Tgfβ treatment in mammary cancer cell lines.

A. Cells were oligofected with mock or Ring1B siRNA and 24 hours later treated with 2 ng/ml Tgfβ. 48 h after transfection, protein and mRNA levels of Fak were determined by western blot and qRT-PCR, respectively. Effectiveness of Ring1B downregulation was confirmed by western blot (upper panel) and qRT-PCR (Supplementary Figure 1). α-Tubulin and Lamin B, loading controls. *, P < 0.05; **, P < 0.005. B. Erk phosphorylation in Ring1B-depleted cells upon Tgfβ treatment. MCF7 and MDA-MB-231 cells were treated as above and 48 hours later p-Erk 1/2 levels were determined by western blot. Erk 2, loading control. C. Subcellular localization of Fak in Ring1B depleted cells upon Tgfβ treatment, monitored by immunofluorescence. Arrows indicate focal adhesions. Bar, 75 μm.