Orthovanadate activates JAK2 and STAT5 but not EPOR.

(A) HCD57 cells were deprived of EPO and cultured 24 hours in 0 (lanes i and ii), 10 (lanes iii and iv), or 70 (lanes v and vi) μmol/L orthovanadate. The cells were either stimulated with 10 units EPO per milliliter (lanes ii, iv, and vi) or mock treated for 5 minutes (lanes i, iii, and v). Cell extracts were then immunoprecipitated with anti-EPOR antibody. Immunoprecipitates were analyzed by Western blot (WB) with antiphosphotyrosine antibody. The blot was stripped and reprobed with anti-EPOR antibody (lower panel). (B) HCD57 cells were deprived of EPO and cultured 24 hours in 0 (lanes i and ii), 10 (lanes iii and iv) or 70 (lanes v and vi) μmol/L orthovanadate. The cells were either stimulated with 10 units EPO per milliliter (lanes ii, iv, and vi) or mock treated for 5 minutes (lanes i, iii, and v). Cell extracts were then immunoprecipitated with anti-JAK2 antibody. Immunoprecipitates were analyzed by Western blot with antiphosphotyrosine antibody. The blot was stripped and reprobed with anti-JAK2 antibody (lower panel). (C) HCD57 cells were deprived of EPO and cultured 24 hours in 0, 10, or 70 μmol/L orthovanadate. The cells then were either stimulated with 10 units EPO per milliliter or mock treated for 5 minutes. Nuclear extracts were analyzed by electrophoretic mobility shift assay (EMSA) with the STAT5 DNA binding sequence (PIE) for STAT5a/b DNA binding activity (lanes i-vi). Supershifts were performed with anti-STAT5a (lanes vii and ix) or anti-STAT5b (lanes viii and x) antibodies.