Figure 6. Rabbit A3A forms heterodimers and is negatively regulated by rabbit TRIB3.

(A) Representative FRET FACS assay of HEK-293T cells 48 hours post transfection with rA3A-Cherry expression plasmid in combination with an empty vector (ev), rA3A-GFP, or rA1-GFP expression plasmid. The percentage of FRET⁺ cells were gated on Cherry⁺ cells for single transfection or Cherry⁺ and GFP⁺ cells for co-transfection. (B) Confocal microscopy analysis of V5-tagged APOBEC and 3xFLAG-tagged TRIB3 rabbit proteins in HeLa cells 24 hours post co-transfection. Nuclei are stained using DAPI. (C) Representative FRET-FACS assay of HEK293T cells 48 hours after transfection with rabbit APOBEC-Cherry or TRIB3-GFP constructs alone or in combination. A plasmid encoding a Cherry protein fused to a GFP protein was used as a positive control. Percentage of FRET⁺ cells are gated on Cherry⁺ cells for single transfection or Cherry⁺ and GFP⁺ cells for co-transfections. (D) Western blot detection of V5-tagged rA3A and 3×FLAG-tagged rTRIB3 proteins in HEK-293T transfected cells by either rA3A alone or rA3A + rTRIB3 (1/4 molar ratio). β actin was used as a loading control. Quantification of fluorescence intensity was performed using ImageJ by normalizing datas to rA3A, D.R, densitometric ratio. (E) Human CMYC DNA specific 3D-PCR gels from HEK293T-UGI cells 48 hours post transfection with either an empty vector (ev), rA3A alone and rA3A and rTRIB3 together (1/4 ratio). Numbers above the gel indicates the Td for each well. White bar indicates restrictive Td. (F) FACS analysis of HeLa cells 48 hours after transfection with an empty vector (ev), rTRIB3, rA3A and rA3A and rTRIB3 together (1/4 ratio). Percentage of γH2AX⁺ cells are gated on V5-Tag positive cells except for mock and rTRIB3 conditions. A single asterisk means the difference is statistically significant (p ≤ 0.05) and a double asterisk means the difference is highly statistically significant (p ≤ 0.005).