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Fig. 7. DLG1 phospho-specific antibodies specifically recognise DLG1 phosphorylated by CDK1 and CDK2. (A,B) GST-DLG1 was either phosphorylated (+) or not (–) in vitro by CDK2 (A) and CDK1 (B), and then subjected to western blot analysis with anti-DLG1 monoclonal antibody (WB DLG1), the anti-Ser442-P and anti-Ser158-P antibodies. The lower panel shows the ponceau stain of the nitrocellulose membrane used for the CDK1 analysis and demonstrates equal levels of protein loading in all lanes. Note equal levels of reactivity with the anti-DLG1 antibody and specific recognition of the phosphorylated forms of DLG1 by the anti-phospho antibodies. (C) HaCaT cells were synchronised with aphidicolin and then released and cell extracts harvested at different phases of the cell cycle. DLG1 was then immunoprecipitated with anti-DLG1 monoclonal antibody, and the pattern of DLG1 expression analysed by western blotting with anti-DLG1 monoclonal antibody (WB DLG1) and the anti-Ser158-P and the anti-Ser442-P antibodies. Cell extracts were also analysed using anti-tubulin antibody to confirm equal amounts of protein extraction. Note low level of reactivity of the anti-phospho antibodies in asynchronous (Asyn) and G1-arrested cells and the specific increases in S (5 hours for Ser158, 6.5 hours for Ser442) and M phases (9 hours for Ser158) of the cell cycle. (D) Asynchronously growing HaCaT cells were untreated (–) or treated (+) with roscovitine (50 µM) for 3 hours. DLG1 was then immunoprecipitated with anti-DLG1 monoclonal antibody, and the pattern of DLG1 expression analysed by western blotting with anti-Dlg monoclonal antibody (WB Dlg) and the anti-Ser158-P antibody. Cell extracts were also analysed using anti-tubulin antibody to confirm equal amounts of protein extraction. Note the loss in reactivity of the anti-phospho antibody treatment.
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