Fig. 1.

The Rsu1 is necessary for activation of p38 Mapk and MKK4 but not MKK3/6 in response to EGF. (a, b) Cell lysates were isolated from control siRNA and Rsu1 siRNA transfected MCF10A cells following EGF treatment (100 ng/ml) for the indicated time. Immunoblot analysis was performed with equal amounts of protein samples as indicated in Materials and Methods and the levels of phospho-p38 (T180/Y182), phospho-MKK4 (S257 and T261), phospho-MKK3/6 (S189/S207), phospho-JNK (T183/Y185), were detected. Total p38, JNK, MKK4 and MKK3/6 were measured and α-tubulin and Rsu1 antibodies were used for loading controls and depletion levels, respectively. c MCF10A cells stably expressing control vector (pBabe), Rsu1-myc (Rsu1) or Rsu1-N92D-myc (Rsu1-N92D) were transfected with negative control siRNA or endogenous Rsu1 specific siRNA and treated with EGF (100 ng/ml) for 10 min as described in Materials and Methods. The lysates were subjected by western blot and analyzed with phosphorylation specific antibodies indicated. d Lysates from MCF10A cells depleted of MKK4, MKK3 or MKK6 were examined for levels of phospho-p38, phospho-JNK and phospho-Erk. Quantitation is expressed as fold stimulation compared to T = 0 for control siRNA transfected cells set as 1