Prolyl hydroxylase enzyme is up-regulated and active in CLL B cells. (A) Constitutive levels of HIF-1α up-regulates PHD2 levels. Lysates prepared from CLL B cells (P1-6), normal PBMCs (N1), and purified normal B cells (N2) were analyzed for the expression of PHD2, a downstream target of HIF-1α, by Western blot using a specific antibody. Constitutive levels of HIF-1α in these samples are also shown. Actin was used as the loading control. (B) CLL B cells express heavily hydroxylated HIF-1α. CLL B-cell lysates (labeled as P7-9) were subjected to immunoprecipitation using a monoclonal antibody to HIF-1α. Immunoprecipitated protein was analyzed for posttranslational modification by Western blot using a polyclonal praline (564)–specific hydroxylated HIF-1α antibody. The blot was stripped and reprobed to detect immunoprecipitated HIF-1α. Raji cells pretreated with 100 μM CoCl2 for 6 hours were used as a negative control.
