PU-H71 recognizes Hsp90 bound to the vFLIP signalosome and shows preferential binding to the long form of cFLIP. (A) Immunoblot from PU-H71 pull-downs, but not control bead pull-downs, using extracts from 5 different KSHV+ PEL cell lines (BC3, BC1, BC2, BC5, and BCBL1), recognized vFLIP and IKKγ. Control extracts from a B-cell lymphoma cell line lacking KSHV and thereby vFLIP (IBL1) showed binding of Hsp90 to IKKγ, but no bands were seen with the vFLIP antibodies, indicating specificity of the vFLIP band. Results shown are representative of 2 independent experiments. (B) Immunoblots from PU-H71 pull-downs were evaluated for Hsp90 binding to cFLIP, the cellular homolog of vFLIP. Although vFLIP is more closely structurally related to the short form (cFLIPS), PU-H71 failed to recognize this protein, and only the long form (cFLIPL) was identified. (C) Schematic representation of cFLIP long and short forms is shown; the most active of these proteins in terms of ability to activate NF-κB is vFLIP, followed by cFLIPL and the by cFLIPS.21,23
