Fig. 4. Activation of MAPK and PI 3-K by NRG1 and NRG2. Cells were stimulated with NRG1 or NRG2 and some cultures were preincubated for 1 hour with PD98059 (40 µM) or LY294002 (4 µM), or for 10 hours with PG-J2. (A) PI 3-K activation was determined by measuring the extent of Akt phosphorylation by western blotting using an anti-phospho-Akt-specific goat polyclonal antibody. Total Akt protein was determined by stripping and reprobing the blot with an anti-Akt polyclonal antibody. (B) MAPK activation was determined by measuring the extent of MAPK phosphorylation by western blotting using an antiphospho-MAPK (p42 and p44)-specific mouse monoclonal antibody. Total MAPK protein content was determined by stripping and reprobing the blot using an anti-MAPK-p42 and an anti-MAPK-p44 polyclonal antibody.