Fig. 4
- ID
- ZDB-FIG-250204-27
- Publication
- Sun et al., 2024 - Target protein identification in live cells and organisms with a non-diffusive proximity tagging system
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Dasatinib-HTL (DH) derivatives with modified and longer linkers enhances POST-IT performance. (A) In vitro binding assay for DH derivatives via fluorescence polarization (FP) assay. Purified Myc-Halo-PafA (15 nM) and 1 nM Halo-AF488 were incubated with various DH derivatives in a serial dilution at 37°C for 30 min prior to FP measurement. n = 2. Data are shown as mean ± s.d. (B) Cellular binding assay for DH derivatives by FP measurement. HEK293T cells transfected with Myc-Halo-PafA were later incubated with 1 μM of each DH derivative or DMSO for 3 hr, followed by cell lysate incubation with 2 nM of Halo-AF488 at 37°C for 30 min. n = 4. Data are shown as mean ± s.e.m. p-Values were calculated by an unpaired two-sided t-test. *p<0.05; **p<0.01; ***p<0.001; versus DMSO. Immunoblot (IB) presents a representative image of the input levels for each condition, demonstrating that treatment with DH derivatives did not alter the expression levels of Myc-Halo-PafA. (C) In vitro labeling activity comparison among DH derivatives. Purified recombinant Halo8KR-PafA (0.5 μM), SBPK4R-sPupK61R (10 μM), and SRC(247-536)-2xV5 (0.5 μM) were incubated with 0.5 μM of each DH derivative at 37°C for 30 min. (D, E) In cellular labeling activity comparison among DH derivatives. HA-Halo8KR-PafA and SBPK4R-sPupK61R were used for co-transfection, and cells were treated with 250 nM of various DH derivatives. Pupylation levels of purified short SRC (C), endogenous SRC (D), or pull-downed endogenous SRC (E) were assessed by IB using anti-V5 or anti-SRC antibodies for exogenous or endogenous SRC, respectively. |