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. 2005 Nov 22;392(Pt 2):389–397. doi: 10.1042/BJ20042102

Figure 3. Pcp2 expression stimulates Ras but not ERK1/2.

Figure 3

(A) Basal Ras activation +/−Pcp2 expression. PC12 cells were cultured +/−Dox for 48 h and Ras activity was determined from 500 μg of cell lysates as described in Experimental section. Western blot for Ras detected after elution from Raf-1-RBD GST agarose (active Ras) and total Ras (from 50 μg cell lysate). In parallel, Dox+lysates were incubated with GTP[S] as a positive control and GDP as a negative control as described in the Experimental section. (B) Relative Ras activation in Pcp2 expressing cells (−Dox) compared with cells without Pcp2 expression (+Dox). The increase was 5.4-fold ±1.6 from four independent experiments. (C) Ras activation with NGF stimulation (0–30 min) after overnight serum starvation. Relative Ras activation in the presence of NGF is compared in (D) (n=4 for 5 min; n=2 for 15 and 30 min and are expressed ±S.D. at 5 min ± range for 15 and 30 min). Lines separate specific lanes from the same gel and exposure. (E) Western blot of basal- and NGF-stimulated total and phospho-ERK1/2 activity on +/−Dox treated cell lysates. (F) Relative phospho-ERK1/2 (means±S.D. of three experiments).