FIG. 7.

Cytosolic and centrosomal forms of CPAP are associated with γ-tubulin. (A) CPAP and γ-tubulin are present in the centrosomes. The centrosome fractions of Molt4 cells were prepared by discontinuous sucrose density gradient (70, 50, and 40% sucrose solutions). Gradient fractions were immunoblotted with either anti-CPAP (upper) or anti-γ-tubulin (lower) antibodies. (B) CPAP and γ-tubulin cosedimented in the same cytosolic fractions. The cytosolic fractions prepared from Molt4 cells were loaded on a 15 to 40% sucrose linear gradient as described in the text. After centrifugation, the fractions were immunoprobed with anti-CPAP and anti-γ-tubulin antibodies. The lane marked T represents a positive control by immunoblotting of the Molt4 cell cytosolic extracts with anti-CPAP and anti-γ-tubulin antibodies. (C) Colocalization of CPAP and γ-tubulin in the isolated centrosomes. The centrosome fractions in panel A were spun onto acid-treated coverslips for immunofluorescence analysis. The coverslips were then double stained with anti-CPAP (left) and anti-γ-tubulin (right) antibodies. Bar, 10 μm. (D) Coimmunoprecipitation of CPAP and γ-tubulin in vivo. The cell extracts of SiHa cells were immunoprecipitated (IP) with NRIgG (lane 1) or anti-CPAP (lane 2) antibody. The bound protein complexes were analyzed by immunoblotting (IB) with anti-γ-tubulin antibody. To further confirm the association between CPAP and γ-tubulin, the cell lysates were first immunoprecipitated with anti-γ-tubulin MAb (lane 4) or a control hybridoma (H25B10, lane 3). The immunoprecipitated complexes were then reprobed with anti-CPAP antibody. Both results showed that CPAP and γ-tubulin associate in vivo.