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. 2021 Nov 9;95(23):e01323-21. doi: 10.1128/JVI.01323-21

FIG 4.

FIG 4

Evaluation of Gag and GagPol dimerization using FRET assay. HEK293T cells were cotransfected with pGag-EGFP and pGag-mSB (a), pGagPol(WT)-EGFP and pGagPol(WT)-mSB (b), or pGagPol(IN:M50I)-EGFP and pGagPol (IN:M50I)-mSB (c), and then FRET assays were conducted as described in Materials and Methods. Gag-EGFP and Gag-mSB are displayed G-EGFP and G-mSB. GagPol(WT)-EGFP, GagPol(WT)-mSB, GagPol(IN:M50I)-GFP, and GagPol(IN:M50I)-mSB are depicted GP(WT)-EGFP, GP(WT)-mSB, GP(M50I)-EGFP, and GP(M50I)-mSB, respectively. (d) FRET efficiencies were calculated as described in Materials and Methods. Gag and GagPol homodimerization are described as G-G and GP-GP, respectively. The efficiencies of GP-GP of WT and M50I were compared with that of the G-G pair. Data indicate means ± SE from five independent assays. *, P < 0.05; ***, P < 0.001. (e to g) HEK293T cells were transfected with pGag-GFP (a), pGagPol(WT)-GFP (b), or pGagPol(IN:M50I)-GFP (c) construct and the protein distribution was observed. After transfection, cells were fixed with a fixation buffer (Abcam), and tubulins were stained with Alexa Fluor 555 anti-beta tubulin antibody (EPR16774) (ab206627). Scale bars indicate 20 μm.