Stability analysis of PBase protein, facilitated by flow cytometry.
Hela cells were transfected with pTriEx-mPB-2A-eGFP and pTriEx-NP-mPB-2A-eGFP plasmids. (A) APC fluorescence via anti-His tag antibody staining indicated the presence of mPB or NP-mPB. Because the eGFP moiety was cotranslated with PBase variants, the GFP fluorescence intensity was used as a reference for the mPB and NP-mPB translation levels of successfully transfected cells. (B and C) Y-axis represents accumulated cell counts. Although the NP-mPB group had a much smaller percentage of cells that were APC+ (4.65%) than the mPB group (18.2%), the percentages of GFP+ cells were similar (26.7% vs. 32.7%). (D) The APC fluorescence intensity in GFP+ populations represented the protein stability. The ratio of mean APC fluorescence (a.u.) of NP-mPB to mPB was 302 to 671.