Figure 1.
Schematic diagram of the multiplexed Alpha assay for STAT3- and STAT5-SH2 binding.
Figure 2.
STAT3 and STAT5b protein expression.
(A) Western blot analysis of the soluble (sup) and insoluble (ppt) fractions of E. coli expressing STAT5b.The proteins (5 µg) were analyzed by SDS-PAGE, and then probed with an antibody. The arrows indicate the expressed proteins. (B) CBB staining of the STAT3(136–705) and STAT5b(136–703) proteins. The purified soluble proteins (0.5 µg) were analyzed by SDS-PAGE. (C) Western blot analysis of the STAT3(136–705) and STAT5b(136–703) proteins. The blotted proteins were detected with an antibody.
Figure 3.
Effects of the spacer length in digoxygenin (DIG)-labeled GpYLPQTV peptides on STAT3 binding.
(A) Chemical structures of the peptides. DIG-labeled peptides that contain two carbon (C2) or six carbon (C6) spacers were used. (B) Dose dependence of the DIG-C2-GpYLPQTV and DIG-C6-GpYLPQTV peptides on STAT3 binding. Each point represents the mean from three replicates, and the error bars represent the standard deviation from the mean. The signals from 1.0 nM DIG-C6-GpYLPQTV were used as 100%.
Figure 4.
Inhibitory effects of the non-labeled peptides on STAT3- and STAT5b-SH2 binding.
(A) Dose-dependent inhibition of STAT3 binding in the single assay. (B) Dose-dependent inhibition of STAT5b binding in the single assay. (C) Dose-dependent inhibition of STAT3 binding in the multiplexed assay. (D) Dose-dependent inhibition of STAT5b binding in the multiplexed assay. Each point represents the mean from three replicates, and the error bars represent the standard deviation from the mean.
Table 1.
Determination of the Z’ values by the multiplexed assay.
Figure 5.
Identification of Compound 1 and its inhibition of STAT3- and STAT5b-SH2 binding.
(A) Chemical structure of Compound 1. (B) Dose-dependent inhibition of Compound 1 in the single assay. (C) Dose-dependent inhibition of Compound 1 in the multiplexed assay. Each point represents the mean from three replicates, and the error bars represent the standard deviation from the mean.
Figure 6.
Inhibition of the STAT3 nuclear translocation by Compound 1 in HeLa cells.
(A) Fluorescence images of the STAT3 proteins in the nuclei of HeLa cells. The HeLa cells were pretreated with or without the test compounds, and then either treated with oncostatin M or left untreated. The cell nuclei were stained with Hoechst 33342 and the STAT3 proteins were visualized with anti-STAT3 antibody. The merged image showed the fusion of blue (nuclei) and green (STAT3) fluorescence. The test compounds treated are shown in the figure. The cells treated with neither oncostatin M nor the test compounds are shown as “–oncostatin M”. (B) Dose-dependent inhibition of the STAT3 nuclear translocation by Compound 1. The mean fluorescent values of STAT3 in the nuclei were calculated. The signals with oncostatin M but not the test compounds (“vehicle”) and “–oncostatin M” were used for the 100% and 0% signals, respectively. The relative signal intensity was calculated in each well. Each point represents the mean from three replicates, and the error bars represent the standard deviation from the mean (*P<0.05 and **P<0.01 versus vehicle in Student’s t-test). JAK inhibitor 1 was used as a positive control.
Table 2.
Structure-activity relationships for the Compound 1 analogs for the STAT3- and STAT5b-SH2 bindings.