Fig. 6. Stimulation of the P2Y₁R transactivates the EGFR pathway. (A) 2-MeSADP increases the EGFR tyrosine phosphorylation in a concentration-dependent manner. Quiescent FRT and HeLa cells were treated for 5 minutes with either 1 or 10 nM 2-MeSADP or 1 nM EGF, as indicated. The EGFR was then immunoprecipitated, resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine monoclonal antibody 4G10 to detect the tyrosine phosphorylated EGFR (EGFR-P). After stripping, blotted membranes were incubated with polyclonal antibody EGFR984 for total EGFR detection. EGFR-P is clearly more intense than in control cells (`C') without agonist stimulation. (B) 2-MeSADP increases ERK1/2 activation. Quiescent cells treated for the indicated time periods either with carrier culture medium alone (Control, `C') or with 10 nM 2-MeSADP or 1 nM EGF were analyzed for ERK activation by immunoblot against total ERK or phosphorylated ERK (ERK-P). Each graph corresponds to quantification of three independent experiments, normalized with respect to the corresponding total EGFR or ERK protein in each lane. Values are the means ± s.e.m. ^*P<0.05, ^**P<0.01; Dunnett's test compared with control conditions without agonist.