ERK activation in SS RBCs involves the cAMP/PKA pathway and the tyrosine kinase p72^syk and is sensitive to the effect of Gα_i protein. SS RBCs (A-D), and reticulocyte-enriched and -depleted (mature) SS RBCs (E) were sham-treated, treated with forskolin (A), epi (B-C), PKAI, 14-22 amide, (B), or PTx (C-D) in the presence or absence of the MEKI U0126 (A,B,D), piceatannol (D) or damnacanthal (D). RBC proteins were blotted with antibodies against ERK and phosphoERK. Quantitative analysis of the blots is presented as fold change in ERK phosphorylation. (A-B) ERK undergoes phosphorylation via the cAMP/PKA pathway. (A) ERK undergoes increased phosphorylation after RBC incubation with forskolin, which is inhibited by U0126 (n = 3). *P < .05 compared with untreated cells. **P < .01 compared with forskolin-treated SS RBCs. (B) Phosphorylation of ERK is increased by epi, and this increase was abrogated by either 14-22 amide or U0126 (n = 3). *P < .01 compared with untreated cells. **P < .01 compared with epi-treated SS RBCs. (C) ERK phosphorylation in SS RBCs is enhanced by inactivation of the Gα_i protein. PTx at either 1 or 2 μg/mL increased basal ERK phosphorylation (n = 9). *P < .001 compared with nontreated cells; †P < .05 compared with epi-treated SS RBCs. (D) The tyrosine kinase p72^syk is implicated in ERK phosphorylation. PTx at 2 μg/mL up-regulated ERK phosphorylation, an effect that was blocked by piceatannol. Conversely, damnacanthal failed to block ERK phosphorylation induced by PTx (n = 3). *P < .01 compared with untreated cells. **P < .01 compared with PTx-treated SS RBCs. (E) ERK1/2 is phosphorylated at baseline in both reticulocyte-enriched and reticulocyte-depleted (mature) SS RBCs (n = 2).