Fig. 4: CDK6 phosphorylates CAD at S1900.

A 293 T cells were transfected with plasmids expressing Flag-CDK6 and V5-CAD for 48 h. WCLs were precipitated with anti-Flag (CDK6). Precipitated proteins and WCLs were analyzed by immunoblotting with the indicated antibodies. B 293 T cells were transfected with plasmids expressing Flag-CDK6 with or without vCyclin for 48 h. WCLs were precipitated with anti-Flag (CDK6). Precipitated proteins and WCLs were analyzed by immunoblotting with the indicated antibodies. C 293 T cells were transfected with a plasmid expressing Flag-CDK6 for 48 h. WCLs were prepared and analyzed by 2DGE and immunoblotting. D In vitro [γ-³²P]-ATP kinase assay was performed on CAD with purified CDK4 or CDK6 and visualized by autoradiography (Top). The membrane was then stained with Ponceau S (Bottom). Palbociclib: a CDK4/CDK6 inhibitor. E The m/z spectra of the peptide containing S1900 phosphorylation was shown (Left), with its abundance in 293 T cells ectopically expressing an empty vector, vCyclin, or CDK6 (Right). F In vitro ATP kinase assay was performed on CAD wild-type (WT) and S1900A mutant with purified CDK6, which was then analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. G 293 T cells were transfected with a plasmid expressing Flag-CAD and then infected with KSHV-wild-type (WT) or KSHV-vCyclin.STOP (∆Cyclin) (MOI = 5) for 24 h. WCLs were precipitated with anti-Flag (CAD). Precipitated proteins and WCLs were processed in parallel and analyzed by immunoblotting with the indicated antibodies. H 293 T cells were depleted of CDK6 by CDK6-speific shRNA and then infected with KSHV (MOI = 5) for 24 h. WCLs were precipitated with anti-CAD. Precipitated proteins and WCLs were processed in parallel and analyzed by immunoblotting with the indicated antibodies. Data are presented as mean ± SD of n = 4 biological replicates (4E). Blots were representative of at least two independent experiments (4A-4D, 4F-4H). Source data are provided as a source data file. See also Fig. S4.