Fig. 4

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.

Expression Data

Expression Detail
Antibody Labeling
Phenotype Data

Phenotype Detail
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Elife