ERK1/2 signaling up-regulates glycophorin A serine phosphorylation. Inorganic 32P radiolabeled intact SS RBCs were incubated in the absence (patient 1: lane 1) or presence (patients 1: lanes 2, 3 and 4; and patient 2: lanes 1, 2 and 3) of serine/threonine protein phosphatase inhibitors (SPI), not followed (patient 1: lanes 1 and 2; and patient 2: lane 1) or followed by treatment with epinephrine (epi) (patient 1: lanes 3 and 4; and patient 2: lanes 2 and 3). In lane 4 (patient 1) and lane 3 (patient 2), SS RBCs were preincubated with MEK1/2 inhibitor U0126 prior to treatment with SPI followed by epi treatment. The Fold change in phosphorylation is representative of three different experiments, calculated after subtraction of cpm present in a lane (not shown) containing immunoprecipitates using immunoglobulin P3 from cpm obtained using anti-glycophorin A mAb for immunoprecipitation under each set of conditions indicated. *: p<0.05 and p<0.001 for SPI-treated and SPI+epi-treated vs. sham-treated, respectively; **: p<0.001 compared to SPI+epi-treated SS RBCs. Total glycophorin A loaded in each lane was detected using nitrocellulose membranes of phosphorylated glycophorin A blotted with anti-glycophorin A mAb.