ERK signaling modulates both SS RBC adhesion to endothelial cells and ICAM-4 phosphorylation. (A-B) Activation of ERK signaling up-regulates SS RBC adhesion to the endothelium. SS RBCs were sham-treated, stimulated with epi for 1 minute or forskolin, preincubated with U0126 followed by epi or forskolin, or treated with U0126 alone. Adhesion of SS RBCs to HUVECs was tested in intermittent flow condition assays. Results are presented as percent adherent SS RBCs at a shear stress of 2 dynes/cm². Error bars show SEM of 4 different experiments. (A) *P < .001 compared with sham-treated; **P < .001 compared with epi-treated. (B) *P < .001 compared with sham-treated; **P < .001 compared with forskolin-treated. (C-D) The MEK/ERK signaling cascade is involved in ICAM-4 (LW) serine phosphorylation. (C) Inorganic ³²P-radiolabeled intact SS RBCs were incubated in the absence (lane 1) or presence (lanes 2, 3, 4, 5, and 6) of serine/threonine protein phosphatase inhibitors (SPI), followed or not (lanes 1 and 2) by treatment with epi (lanes 3, 4, 5, and 6). In lanes 4, 5, and 6, SS RBCs were preincubated with SPI in presence of PKAI, MEKI, or PKAI + MEKI followed by epi treatment, respectively. The counts per minute (cpm) are representative of 3 different experiments, calculated by subtraction of cpm present in a lane (not shown) containing immunoprecipitates using immunoglobulin P3 from cpm obtained using anti-LW (ICAM-4) mAb for immunoprecipitation under each set of conditions indicated. *P < .05 and *P < .001 for SPI-treated and SPI + epi-treated vs sham-treated, respectively; **P < .001 compared with SPI + epi-treated SS RBCs. Total LW loaded in each lane was detected with the use of nitrocellulose membranes of phosphorylated LW blotted with anti-LW mAb. (D). SS RBCs were incubated without (lanes 1 and 3) or with epi (lanes 2 and 4). Lanes 1 and 2 were immunoprecipitated with P3. Lanes 3 and 4 were immunoprecipitated with anti-LW mAb; all lanes for panel D were immunostained with anti-LW mAb.