Fig. 4. Cell cycle-dependent PCTAIRE-1 kinase activity and tyrosine phosphorylation in Hs68 fibroblasts. Hs68 cells were made quiescent by serum deprivation (48 h) and restimulated with 10% FCS for 20 h. A, cell cycle progression was monitored by flow cytometric analysis as described in "Materials and Methods." B, extracts made from synchronized Hs68 fibroblasts (200 µg) at different times during the transition through the cell division cycle were immunoprecipitated and assayed for kinase activity as described in "Materials and Methods." G0 (Lane 1), early G1 (2 h; Lane 2), late G1 (8 h; Lane 3), G1-S phase (Lane 4), S phase (Lane 5) and G2 (Lane 6) phase extracts were immunoprecipitated with anti-PCTAIRE-1 antibody. S-phase extracts treated with no antibody (Lane 7) or immunoprecipitated with anti-cyclin A antibody (Lane 8) were used as negative and positive controls, respectively. The results of an experiment representative of three independent determinations are shown. C, an aliquot (15 µg) of the extracts used in A was probed with PCTAIRE-1 antibody to examine the level of endogenous PCTAIRE-1 expression during cell cycle progression. D, extracts (200 µg) made in G1 or upon release from the hydroxyurea block (see "Materials and Methods") were immunoprecipitated using a monoclonal antiphosphotyrosine antibody. Proteins were resolved by SDS-PAGE and transferred to PVDF membrane. The membrane was probed with an anti-PCTAIRE-1 antibody and revealed with the ECL detection kit. G1 (Lane 1) or hydroxyurea block-released (Lane 2) Hs68 cells. G1 extract treated with no antibody was used as a negative control (Lane 3). The band below PCTAIRE-1 is nonspecifically detected by the antibody used to probe the membrane because it is also present in the negative control (Lane 3).