Figure 4
From: Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags
Application of GPI-epitope tags for targeting of essential genes. (a) Schematic illustration of constructs generated to encode for monomeric GFP mClover which is N- or C-terminally fused to smAID and CD5HA2 separated by ribosome skipping sequence P2A. (b) FACS plots showing 293 T cells stained for surface HA-tag. Cells were transiently cotransfected with one of the targeting plasmids and either osTIR1 expression vector or mock plasmid for 24 h and then treated with 0.5 µM auxin for 16 h. (c) WB analysis of native and C-terminally tagged Ku70 expressed in 293 T cells after KI and selection with one or two GPI-epitope tags. Flag+HA+ cells were also stably transduced with pUCHR-osTIR1-IRES-GFPt vector and treated with auxin for 16 h. (d) Dynamics of Ku70 degradation tagged with smAID. Cells (lane 5 in c) were incubated with auxin for the indicated periods of time, lysed and analyzed by WB. Images are representative of at least two experiments; the uncropped blots are shown in Supplementary Fig. 8.
