Figure 4.

SpyTag/SpyCatcher can improve the stability of Split-GFP.A, schematic representation of the Spy-GNCre_{aa 59} (NCre_{aa 19–59}-NGFP_1–7-SpyTag) and Spy-GCCre_{aa 60} (SpyCatcher-CGFP_8–11-CCre_{aa 60–343}) fusion proteins in this study. All constructs were inserted into the pMSCV-Thy1.1 expression reporter vector. B, HEK 293T cells were transiently transfected with components of the Split-GFP-Cre or Split-Spy-GCre system. The cells were harvested 20 h after transfection, and cells with different Cre expression levels as indicated by the Thy1.1 expression level were analyzed by flow cytometry. C, the fluorescence intensity of GFP among cells expressing different levels of Cre (Thy1.1 low, medium, and high) is shown. HEK 293T, human embryonic kidney cancer cells 293T; ns, not significant. ∗∗∗p < 0.001 (ANOVA with Bonferroni post hoc test).