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. 2000 Sep;11(9):2863–2872. doi: 10.1091/mbc.11.9.2863

Figure 1.

Figure 1

PEA-15 augments Ras and Raf activation of ERK2. (A) CHO cells were cotransfected with HA-ERK2 (2 μg) and 3 μg of expression vectors encoding H-RasG12V (H-Ras) or RafCAAX (Raf) in combination with PEA-15 (3 μg) or vector lacking an insert (3 μg). Cells transfected with 6 μg of vector cDNA also were assayed (Control). Transfected ERK2 was immunoprecipitated and its activity was assayed by its ability to phosphorylate myelin basic protein. Autoradiographs of three independent experiments were analyzed by spot densitometry as described under MATERIALS AND METHODS. Cotransfection of Raf and PEA-15 activates ERK significantly better than transfection of Raf alone (p = 0.0), whereas cotransfection of Ras and PEA-15 does not clearly activate ERK significantly better than Ras alone (p = 0.05). Depicted is the ERK2 activity relative to maximal activation (Raf + PEA-15). Note that PEA-15 augmented activation by both RafCAAX and RasG12V. Mean ± SD (n = 3). (B) αβpy-cells were cotransfected with expression vectors encoding 3 μg of RafCAAX or H-RasG12V in combination with PEA-15 (4 μg) or vector lacking an insert (4 μg). After 48 h, integrin activation was assayed by PAC1 binding as described under MATERIALS AND METHODS. Shown is the mean activation index ± SD of three independent experiments.