Fig. 3. Analysis of the vFLIP-IKK complex in a KSHV-infected B-cell line. (A) Cytoplasmic extract from BC3 cells was subjected to gel filtration on a Superose 6 column, then fractions were analysed by immunoblot with anti-IKK, ß or , anti-vFLIP and anti-vCyclin antibodies. Fractions were also immunoprecipitated with the anti-vFLIP antibody 6/14 and IB kinase activity was measured. The elution volume of protein standards is indicated. (B) Cytoplasmic extracts from BC3 cells were immunoprecipitated using the anti-vFLIP antibody or an isotype-matched control. The immune complexes were incubated with wild-type or mutant (S32A/S36A) GST-IB substrates. The lower panel shows a Coomassie stained gel of the GST-IB in each reaction. (C) Cytoplasmic extract from BC3 cells was immunoprecipitated using an anti-IKK antibody. Equivalent proportions of the original extract (Total lysate), the supernatant from the immunoprecipitation (Supernatant) and the immunoprecipitate (IKKIP) were then analysed by immunoblot with anti-vFLIP and anti-IB antibodies.