Orthovanadate maintains Bcl-XL levels and induces BAD phosphorylation.
(A) HCD57 cells were cultured in the presence of EPO (lane i), in the absence of EPO (lanes ii and iii), or in the absence of EPO and supplemented with 10 (lanes iv and v) or 70 μmol/L (lanes vi and vii) orthovanadate for 72 and 96 hours. Cell extracts were then immunoprecipitated with anti–Bcl-XL (Santa Cruz) antibody raised in rabbit. Immunoprecipitates were analyzed by Western blot with anti–Bcl-XL (UBI) antibody raised in mouse. (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 either were stimulated with 10 units EPO/mL (lanes ii, iv, and vi) or mock treated for 5 minutes (lanes i, iii, and v). Whole cell extracts were analyzed by Western blot with anti–anti-phosphoBAD antibody. The blot was stripped and reprobed with anti-BAD antibody.
