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Fig. 4. DLG1 associates in vivo with CDK1 and CDK2. Asynchronously growing HEK293 cells were extracted with E1a buffer and extracts immunoprecipitated with anti-CDK2 (A), anti-cyclin B (B) antibodies (IP) or pre-immune antibody as indicated. Co-precipitated DLG1 was detected by western blot analysis with anti-DLG1 monoclonal antibody (upper panels) and immunoprecipitated CDK2 and cyclin B detected also by western blotting (lower panels). 20% of each cell extract was used as input control. (C) HaCaT cells treated without nocodazole (–) or with nocodazole (+Noco) for 18 hours were thereafter treated with 50 µM roscovitine (+ Rosco) for 3 hours to inhibit CDK1 activity. The pattern of DLG1 expression was then analysed by western blotting with anti-DLG1 monoclonal antibody. Note the decrease in the intensity of the slower migrating forms of DLG1 in both the asynchronous and nocodazole-arrested cells following treatment with roscovitine. (D) The FACS analysis (bottom two panels) confirms M-phase arrest in nocodazole with (+) and without (–) roscovitine treatment, with the upper two panels showing asynchronously growing cells with (+) and without (–) roscovitine treatment.
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