Fig. 3
- ID
- ZDB-FIG-250204-23
- Publication
- Sun et al., 2024 - Target protein identification in live cells and organisms with a non-diffusive proximity tagging system
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POST-IT can label target proteins in live cells. (A) Effect of linker length between Halo8KR and PafA on target-labeling. HEK293T cells were co-transfected with HA-Halo8KR-PafA containing the specified length of linker, SBPK4R-sPupK61R, and SRC(247-536)-2xV5. (B) Introducing mutations S126A and K172R into PafA significantly enhances its proximity-tagging efficiency on target. (C–E) Comparison of labeling activity of Halo-PafA and Halo8KR-PafAS126A,K172R, a form used for POST-IT. (F, G) POST-ITDH1 mediates proximity-tagging in a DH1-dose dependent manner (F), an effect completely inhibited by competitive dasatinib, ranging from 0.025 to 25.6 μM (G). Pupylation levels of exogenously expressed short SRC (C, F, G), endogenous SRC (D), or pull-downed endogenous SRC (E) were assessed by immunoblot (IB) analysis using antibodies against V5-tag or SRC for exogenous or endogenous SRC, respectively. In all experiments, SBPK4R-sPupK61R was used for co-transfection, and cells were treated with 500 nM DH1, except in (F, G) |