Fig. 3 - Supplemental 1
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- ZDB-FIG-250204-24
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- Sun et al., 2024 - Target protein identification in live cells and organisms with a non-diffusive proximity tagging system
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Test of small molecule-induced proximity-tagging by PafA in HEK293T cells. (A) Schematic illustration of plasmid constructs and a model of proximity-tagging by PafA. In the presence of rapamycin, HA-FKBP-EGFP and V5-FRB-mKate2-PafA heterodimerize via rapamycin, facilitating proximity for PafA to pupylate HA-FKBP-EGFP. (B) V5-FKBP-EGFP is highly pupylated only in the presence of rapamycin, while FRB-mKate2-PafA undergoes substantial self-pupylation regardless of rapamycin treatment. (C) The pupylation levels of the target, HA-FKBP-EGFP, and the self-pupylation of V5-FRB-mKate2-PafA increase in an HB-Pup dose-dependent manner. (D) Evaluation of different Pup substrates on proximity-tagging and self-pupylation. (B–D) HEK293T cells were co-transfected with HA-FKBP-EGFP, V5-FRB-mKate2-PafA, and an indicated Pup substrate. Twenty-four hours post-transfection, the cells were treated with 100 nM rapamycin for an additional 24 hr. Pupylation levels of HA-FKBP-EGFP and V5-FRB-mKate2-PafA were assessed by immunoblot (IB) using antibodies against HA-tag and V5-tag, respectively. |