FIG. 2.

p14^ARF activates CHK1/2 kinases to arrest cells in G₂. (A) Expression of CHK1 and CHK2 proteins was studied by Western blotting in H358/Tet-On/p14^ARF cells cultured for 72 h in the presence or absence of doxycycline (Dox) as indicated. Phosphorylation of CHK1 and CHK2 proteins was confirmed using specific phospho-antibodies. (B) The same analysis was performed with MRC5 human fibroblasts transiently transfected with control pcDNA3.1 or pcDNA3.1/p14^ARF vector. (C) Equal amounts of CHK1 or CHK2 protein were immunoprecipitated (IP) from H358/Tet-On/p14^ARF cells and tested for their ability to phosphorylate a GST-Cdc25C fusion protein. Irrelevant mouse or rabbit immunoglobulins (IgG) were used as negative controls. Phosphorylation of GST-Cdc25C at the serine 216 residue was assessed by immunoblotting using a specific phospho-antibody. GST or GST-Cdc25C recombinant proteins were detected by immunoblotting using an anti-GST antibody (arrows). (D) H358/Tet-On/p14^ARF cells were transfected for 72 h with either mismatch or specific chk1 and chk2 siRNAs as indicated and subjected to Western blot (left panel) and cell cycle (right panel) analyses. Apoptosis was detected by immunoblotting with an anti-active caspase 3 antibody.