LW undergoes serine phosphorylation. (A-B) Inorganic 32P-radiolabeled intact SS RBCs (A) and normal RBCs (B) were incubated in the absence (lanes 1,3) or presence (lanes 2,4) of Ser/Thr protein phosphatase inhibitors (SPIs), followed by treatment either without (lanes 1-2) or with 80 μM forskolin (lanes 3-4). (A, lane 5) SS RBCs were first incubated with 30 nM PKAI, followed by treatment with 80 μM forskolin and SPIs. The cpm shown are the cpm representative of 1 of 3 experiments, calculated by subtraction of cpm present in a lane (not shown) containing immunoprecipitate obtained using negative control P3 rather than anti-LW mAb from cpm obtained using anti-LW mAb for immunoprecipitation under each set of conditions indicated. (C-D) SS RBCs (C) and normal RBCs (D) were incubated without (lanes 1,3) or with (lanes 2,4) 80 μM forskolin. Lanes 3-4 were immunoprecipitated with anti-LW mAb, while lanes 1-2 were immunoprecipitated with the negative control immunoglobulin P3; all lanes for both panels C and D were then immunostained with anti-LW.
