Figure 4.

STIL interacts with CPAP and forms a complex with hSAS6. (A) Schematic of interactions between STIL and CPAP. E1235V: a natural mutation of CPAP/CENPJ (E1235V) with glutamate replaced by valine at amino acid 1235 (Bond et al, 2005). (B) STIL interacts with CPAP in transfected cells. HEK 293T cells were co-transfected with Flag–STIL and each GFP-tagged cDNA construct (Plk4, hSAS6, CPAP, STIL, and Cep152). Twenty-four hours after transfection, the cell lysates were first immunoprecipitated (IP) with anti-Flag antibody and then immunoblotted (IB) using anti-GFP and anti-Flag antibodies. (C) Endogenous STIL, CPAP, and hSAS6 form a complex in vivo. Western blot analysis of immunoprecipitated complexes containing endogenous CPAP, STIL, and hSAS6 prepared from HEK 293T cell lysates using anti-CPAP antibody and a non-relevant normal IgG (Mock). (D) The natural E1235V mutation of CPAP reduced its binding to STIL. HEK 293T cells were co-transfected with full-length GFP–CPAP-wt or GFP–CPAP-E1235V mutant with Flag–STIL. Twenty-four hours after transfection, the cell lysates were immunoprecipitated with anti-Flag or anti-GFP antibodies and analysed by immunoblotting using indicated antibodies. (E) Western blot analysis of immunoprecipitated complexes from HEK 293T cells transfected with GFP–CPAP-CP3 (wt, 895–1338) or GFP–CPAP-CP3 (E1235V) mutant. The cell lysates were immunoprecipitated with STIL antibody or a non-relevant normal IgG (Mock) and analysed by western blot using indicated antibodies. (F) GST pulldown assay. GST and GST–STIL (231–619) recombinant proteins were affinity purified and stained with Coomassie blue (C.B. stain, a). Equal amounts of GST and GST–STIL truncated proteins were used to pull down the cell lysates prepared from cells transfected with full-length GFP–CPAP-wt or GFP–CPAP-E1235V mutant constructs. The pulldowns were analysed by western blot using anti-GFP antibody (b).