FIGURE 6.

TRiC/CCT is required for the efficient binding of WDR68 to DYRK1A. COS7 cells were transfected with either control or TCP1α-specific siRNAs along with expression plasmids for 3×FLAG-DYRK1A and HA-WDR68. After 48 h, cell extracts were prepared, and the amounts of DYRK1A (A), WDR68 (B), and TCP1α (C) were examined by Western blotting (WB). Lane 1, no DNA transfection; lane 2, transfected with expression plasmids without siRNA; lanes 3 and 4, two independent control siRNAs; lanes 5 and 6, two independent TCP1α-specific siRNAs (lane 5, HSS110578; lane 6, HSS186251). D and E, the binding of WDR68 to DYRK1A was examined by co-immunoprecipitation experiments (IP), and the amounts of DYRK1A (D) and WDR68 (E) in the DYRK1A immunoprecipitates were examined by Western blotting. Lanes 1–6, same as in A–C; lane N, extract of cells without DNA transfection as a negative control; lane P, extract of cells transfected with DYRK1A and WDR68 as a positive control. F, WDR68 in cell extracts and in the immunoprecipitate was analyzed by neutral pH gradient gel electrophoresis followed by Western blotting. Lanes 1 and 2, control siRNA-transfected cell extracts; lanes 3 and 4, TCP1α-specific siRNA-transfected cell extracts; lane 5, DYRK1A immunoprecipitate from control siRNA-transfected cells. The positions of DYRK1A, WDR68, TCP1α, and molecular weight markers are shown in A–F. An asterisk in B indicates nonspecific bands.