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. 2018 Jun 12;9(45):27809–27822. doi: 10.18632/oncotarget.25593

Figure 4. Rabbit A3A deaminates foreign DNA.

Figure 4

(A) Fluorescence resonance energy transfer assay (FRET)-based in vitro deamination assay performed on FAM-TAMRA coupled oligonucleotides using transfected HEK-293T lysates. rA3A* and hA3A* catalytic mutants transfected cells used as negative controls. Results are expressed in Relative Fluorescence Unit per mg of protein (RFU/mg). A single asterisk represents a statistically significant difference (p < 0.05) and a double asterisk represents a highly statistically significant difference (p ≤ 0.005). (B) FACS analysis of HeLa cells 72 hours post transfection with an empty vector (ev) or rabbit A3A proteins. Percentage of GFP positive cells are gated on A3-V5-Tag positive cells. Single asterisks represent a statistically significant difference (p ≤ 0.05) and double asterisks represent a highly statistically significant difference (p ≤ 0.005). (C) GFP plasmid 3D-PCR gels from QT6 cells, 48 hours post co-transfection with GFP encoding plasmid and either an empty or an APOBEC3 expressing plasmid. Numbers indicates the Td for each well. Vertical white bar indicates restrictive Td. White asterisks denote PCR products that were cloned and sequenced. (D) Analysis of the 5′ nucleotide context of C->T transitions identified in cloned GFP DNA 3D-PCR products. Percentages are represented in blue for rA3A and red for hA3A. Horizontal bars represent expected values. Asterisks show a statistically significant difference (p < 0.05).