ERK undergoes activation in SS but not normal RBCs. (A-B) Fifty micrograms of membrane protein ghosts (SS RBC ghosts, n = 4, lanes: SS1, SS2, SS3, and SS4; and normal RBC ghosts, n = 4, lanes: AA1, AA2, AA3, and AA4) were used per lane. Western blots of protein ghosts were stained with antibodies against ERK1/2, glycophorin C as a loading control, and MEK1/2 (n = 3 for SS RBC ghosts, lanes: SS1, SS2, and SS3; and n = 2 for normal RBC ghosts, lanes: AA1 and AA2). (A) ERK1/2 and MEK1/2 are highly expressed in both SS and normal RBCs and are bound to the RBC plasma membrane. (B) Quantitative analysis of the data (normalized according to glycophorin C expression) presented as relative ERK1/2 expression compared with normal RBCs (P < .05 for SS vs normal RBCs, n = 4 for each). (C-D) Normal RBCs (n = 3, lanes: 1, 2, 3, and 4) and SS RBCs (n = 3, lanes: 5, 6, 7, and 8) were sham-treated (lanes 1 and 5), incubated for 1 minute with 20nM epinephrine (epi; lanes 2 and 6), pretreated with the MEKI, U0126, followed by epi treatment (lanes 4 and 8), or treated with U0126 alone (lanes 3 and 7). Mouse 3T3/A31 fibroblast lysate was used as a positive control (lane 9). One hundred micrograms of SS and normal RBC ghost proteins were used per lane. Western blots were stained with antibodies against ERK and phosphoERK. (C) ERK1/2 is phosphorylated at baseline in SS RBCs and undergoes increased phosphorylation by epi stimulation. ERK in normal RBCs was not phosphorylated and completely failed to undergo increased phosphorylation after epi stimulation. (D) Quantitative analysis of the data is presented as fold change in ERK phosphorylation. *P < .01 compared with untreated cells. **P < .001 compared with epi-treated SS RBCs. (E-F) ERK immunoprecipitated from sham-treated (lanes: 1, 2, 5, and 6) and epi-treated (lanes: 3, 4, 7, 8, 11, 12, 13, and 14) SS RBCs was incubated without MBP (lanes: 1, 3, 5, 7, 11 and 13) or with MBP (lanes: 2, 4, 6, 8, 12, and 14) as a substrate for ERK, with equal protein amounts per assay condition. Commercial active recombinant human ERK2 was incubated without MBP (lanes: 9 and 16) or with MBP (lanes: 10 and 15) as negative and positive controls, respectively. (E) Immunoblots indicate that the activity of ERK is conserved and functional in SS RBCs and epi can intensify its activity. SS RBCs obtained from 4 different patients (SS1, SS2, SS3, and SS4) were tested. (F) Quantitative analysis of the data are presented as fold change in ERK phosphorylation (n = 4). *P = .0286 compared with nontreated cells.