Overexpression of activated H-Ras or Rac-1, but not p110 PI-3K, is sufficient for activation of p38 MAPK in hemopoietic cells.

(A) Baf/3 cells or (B) R6/X cells were electroporated with 5 μg pEGFP-C1 empty vector, and 30 μg (unless otherwise specified) control plasmid (vector), constitutively active p21 Ras (G12 H-Ras), constitutively active Rac-1 (V12 Rac-1), or constitutively active PI-3K (p110*). At 16 hours after electroporation, activation of p38 MAPK or PI-3K was measured using phospho-p38 MAPK (p-p38 MAPK) or phospho-Akt (p-Akt) specific antibodies, respectively. Equivalency of loading was confirmed using antibodies capable of recognizing both the phosphorylated and nonphosphorylated form of p38 MAPK (p38 MAPK), or Akt (Akt). The relative expression levels of constitutively active mutants of Ras and Rac-1 were estimated using a monoclonal antibody specific for an epitope tag from human c-myc (myc) common to both.