FIG. 6.

Spry2 mediates the inhibitory effect of p38α on EGFR signaling in confluent cells. (A) Subconfluent (SubC) and confluent (Conf) WT and p38α^−/− MEFs and p38α^−/− MEFs with p38α added back were analyzed by immunoblotting. (B) Subconfluent (SC) and confluent (Conf) p38α^−/− MEFs were stably transduced with scrambled (C) or Sprouty2-directed (Spry2) shRNAs, and total lysates were analyzed by immunoblotting. (C) Overconfluent cell numbers of p38α^−/− MEFs expressing shRNAs as described for panel B were determined by the MTT assay, reading the absorbance (optical density) at 595 nm (OD₅₉₅). Error bars indicate standard deviation of biological replicates. (D) Confluent WT, p38α^−/−, and Spry2 shRNA-expressing p38α^−/− MEFs were serum starved, treated with EGF, and analyzed for EGFR ubiquitination by immunoprecipitation of the endogenous EGFR followed by immunoblotting. The position of the ubiquitinated EGFR (Ub-EGFR) is indicated with a black arrowhead. Molecular mass markers in kDa are indicated. (E) H-Ras^G12V-induced focus formation assay using WT and p38α^−/− MEFs expressing scrambled (−) or Spry2 shRNA. (F) Cell lysates from confluent (Conf) WT and p38α^−/− MEFs were subjected to cCbl immunoprecipitation and then analyzed by immunoblotting. The immunoprecipitation control was performed with an anti-p27^Kip1 antibody. (G) Subconfluent (SubC) or confluent (Conf) WT MEFs were incubated for 12 h with SB203580 or dimethyl sulfoxide (−) and then analyzed by immunoblotting. (H) NIH 3T3 cells (90% confluence) were transiently transfected with MKK6-DD or empty vector (−), harvested when they were confluent (Conf), and analyzed by immunoblotting. (I) Confluent WT and p38α^−/− MEFs were treated with cycloheximide (CHX) for the indicated times and analyzed by immunoblotting. +, present; −, absent; IP, immunoprecipitated; Ub, ubiquitinated; IgG, immunoglobulin G; P, phospho.