Hsp70 interacts with Tcl1 and protects it from degradation. (A) Equal amounts of Daudi protein extracts were immunoprecipitated with agarose-conjugated mouse immunoglobulin G (IgG) or anti-Hsp70 antibody and then subjected to SDS-PAGE and to immunoblot using antibodies specific for Tcl1 and Hsp70. (B) In the reverse immunoprecipitation, Daudi cell lysates were immunoprecipitated with anti-Tcl1 antibody. The immunoprecipitates were analyzed by immunoblotting with anti-Hsp70 and anti-Tcl1 antibodies. (C) HEK-293A cells were treated with Myr (50μΜ), 17AAG (5μM), and their combination for 24 hours. Phospho-Akt (Ser 473) and Hsp70 were detected. (D) HEK-293A cells were transfected with siRNA for HSP70 or control siRNA (100nM for 48 hours) and TCL1 plasmid. Indicated proteins were detected by Western blotting. S.e. indicates short exposure; and l.e., long exposure. (E) Daudi lymphoma cells were transfected with siRNA for HSP70 or control siRNA-A, a nontargeting siRNA designed as negative control (100nM/48 hours), and treated with CHX (1.5μM/48 hours) where indicated. Total lysates were subjected to SDS-PAGE and immunoblotted for Hsp70 and Tcl1 antibodies. (F) Daudi lymphoma cells were treated with DMSO (as control), Myr (10 and 50μM), and 17AAG (1 and 5μM) for 24 hours. Total lysates were subjected to SDS-PAGE and immunoblotted for Hsp70, Hsp90, and Tcl1 antibodies.