FIG 5.

HSP105 is required for the interaction of PP2A with the destruction complex. (A) SW480 cells were transfected with control or HSP105 siRNA for 72 h and were incubated with 25 μM MG132 for the indicated times (in minutes) prior to harvest. Western blots are shown for the indicated proteins. (B) HEK293T cells were transfected with control or HSP105 siRNA for 72 h. Whole-cell lysates were immunoprecipitated with an anti-axin1 antibody, and Western blots for the indicated proteins are shown. (C) HEK293T, SW403, SW480, and LS1034 cells were transfected with control or HSP105 siRNA for 72 h. Whole-cell lysates were immunoprecipitated with an anti-axin1 antibody, and Western blots for the indicated proteins are shown. (D) HSP105 protein was mixed with axin1 or PPP2CA, immunoprecipitated with anti-axin1 or anti-PPP2CA, and analyzed by Western blotting. (E) PPP2CA was mixed with axin1 in the absence or presence of recombinant HSP105 protein overnight, immunoprecipitated with an anti-axin1 antibody, and analyzed by Western blotting. (F) 293T cells were transfected with control or HSP105 siRNA for 16 h, followed by transfection of a vector control or PPP2CA for 48 h. Cells were treated with 25 μM MG132 for 4 h before harvest. Whole-cell lysates were immunoblotted with the indicated antibodies. (G) 293T cells were transfected with control or HSP105 siRNA for 16 h, followed by transfection of a vector or PPP2CA for 48 h. Cells were then treated with 10 μg/ml cycloheximide (CHX) and were harvested at the times indicated. Membrane β-catenin-depleted whole-cell lysates were immunoblotted with the indicated antibodies.