(A, upper panel) Tyrosine phosphorylation of Jak2 in human erythroid cells stimulated by erythropoietin (10 U/mL). Day-8 cells were lysed by the addition of an equal amount of a buffer containing 2% Triton X-100 before and after exposure to erythropoietin (10 U/mL). Jak2 was immunoprecipitated with specific Jak2 antisera. Immune complexes were resuspended in SDS sample buffer. Tyrosine phosphorylation of Jak2 was detected by 4G10 as described in the Materials and Methods. Bands were visualized by chemiluminescence. (A, lower panel) The same nylon membrane used in A was stripped of the antibody and reprobed for Jak2. Bands were visualized by NBT/BCIP. Lanes are the same as in (A). (B, upper and lower panels) The time course of tyrosine phosphorylation of Jak2. Tyrosine phosphorylation of Jak2 was detected as described in (A). Lane 1, resting erythroid cells. Lanes 2 to 5, 1 minute, 5 minutes, 10 minutes, and 30 minutes after exposure to erythropoietin (10 U/mL). (B, upper and lower panels) The time course of tyrosine phosphorylation of Jak2. Day-8 cells were stimulated with erythropoietin (10 U/mL) for the indicated time. Tyrosine phosphorylation of Jak2 was detected as described in (A). (C, upper and lower panels) The dose response of tyrosine phosphorylation of Jak2. Day-8 cells were stimulated with different concentrations of erythropoietin for 10 minutes. Tyrosine phosphorylation of Jak2 was detected as described in (A). {/ANNT;4224n;;0n;0n}A