Figures & data
Figure 1. siRNA-mediated depletion of Rsu1 blocks activation of MKK4 in MCF10A cells. MCF10A cells transfected with a Rsu1 specific or a negative control siRNA were stimulated with EGF (10ng/ml) at 96 hours post transfection and lysates were harvested and examined by western blotting as described previously.Citation35 Antibodies for the expression of proteins include: phospho-MKK4 Ser257 (Cell Signaling Technology #4514), MKK4 (Santa Cruz Biotechnology #166168), phospho-MKK3 Ser189/MKK6 Ser207 (Cell Signaling Technology #9231) Tubulin (Santa Cruz Biotechnology #8035) was used as loading control.
Figure 2. The absence of Rsu1-PINCH1 binding alters MKK4 and p38 Map kinase activation in MCF10A cells. (Left) Expression of either endogenous Rsu1 protein or wt-Rsu1 in the absence of endogenous protein allows activation of MKK4 and phosphorylation of p38 Map kinase in MCF10A cells. (Middle) In cells that are detached from substrate, or depleted of Rsu1, integrin engagement and focal adhesion formation are reduced or absent. This results in a block in the activation of MKK4, but MCF10A cell detachment leads to the activation of p38 by MKK6. (Right) The reconstitution of Rsu1-depleted cells with N92D-Rsu1 does not completely restore FA formation but it does promote MKK4 activation.
