FIG 6.

(A–F) 293T cells were transfected for 48 h with pcTax and HA-Pim kinases. (A) Cell lysates were fractionated into nuclear and cytoplasmic lysates and probed with anti-Tax, anti-HA, and anti-Cyclin B1 and α-Tubulin, control antibodies. (B) Cell lysates were lysed directly in urea buffer and whole cell lysates were probed with anti-Tax and anti-HA antibodies. Actin served as a loading control. (C–D) Cell lysates were fractionated into soluble and insoluble nuclear and cytoplasmic lysates and probed with anti-Tax, anti-HA, anti-Cyclin B1, and anti-α-Tubulin antibodies. In (D) cell lysates were treated for 7 h with MG132 (10 μM) or leupeptin (100 μM). Long and short exposures of tax are presented. (E–F) 293T cells were transfected for 48 h with pcTax, pcDNA-Pim-1, Pim-2, or Pim-3, and/or HA-tagged K48 (E) or K63 (F). All cells were treated with MG132 (10 μM) for 6 h, prior to collection. Cell lysates were immunoprecipitated overnight with anti-Tax antibody. Due to Pim repression of Tax, prior to Western blot analysis, additional lysate was run for extracts containing Pim kinases. Western blots were performed on immunoprecipitates and lysates with anti-Tax, anti-HA (Ub), and Pim kinase antibodies. Actin served as a loading control. (G) Quantification of K48 and K63 immunoprecipitation. Bands were quantified from several HA-immunoprecipitates and normalized to lysates.