Phosphorylation Status of Thymidine Kinase 1 Following Antiproliferative Drug Treatment Mediates 3′-Deoxy-3′-[18F]-Fluorothymidine Cellular Retention
Figure 4
Verification of phosphorylated amino acid residues on TK1 protein.
Ost TK1− cells were transiently transfected with FLAG-pCMV2 plasmids coding for TK1 variants. A) Ost TK1− transient transfections with FLAG-pCMV2 plasmids coding for the indicated TK1 variants were resolved (10 µg) on a 75 µM MnCl2-phos-tag gels and probed for TK1 expression (1∶500). B) [18F]FLT uptake in Ost TK1− cells transiently transfected with the indicated TK1 constructs. The main figure represents the uptake in control and nocodazole (0.5 µg/ml) treated samples. The y-axis represents mean ± S.E.M. expressed as counts per µg of protein. Significant differences following transient transfection compared to non-transfected cells, and significant changes after nocodazole treatment compared to the untreated sample are highlighted by stars. The inset reports the percentage of radiotracer uptake compared to cells transfected with wild type (WT; 100%) TK1. Significant differences are indicated by stars. C) TK1 expression in transfected cells resolved on 12% SDS-PAGE. β actin was used as loading control.