Figure 3.
Multiple A3B properties are not altered by CDK4. A, in vitro kinase assay of the indicated HA-tagged proteins in the presence of recombinant CDK4-Cyclin D1 and [γ-32P]ATP. The top two panels show immunoblots of the indicated proteins, and the bottom panel shows a representative autoradiograph of the same reaction following separation by SDS-PAGE. B, fluorescence microscopy of 293T cells stably expressing A3B–eGFP following transfection with siCtrl or siCDK4 RNAs (scale bar, 20 μm). Histogram showing CDK4 mRNA levels from RT-qPCR relative to TBP mRNA (TATA-binding protein). C, PAGE analysis of DNA deamination reaction products following incubation of ssDNA substrates with U2OS whole cell extracts ± A3B and CDK4 depletion as indicated (see text and “Experimental procedures” for details). Recombinant A3A and no APOBEC3 provide positive and negative controls, respectively. Immunoblot controls are shown below.