Figure 7. Brevilin A blocks JAKs-JH1 tyrosine kinase activity in vitro.

(A) Purified hSTAT3 protein and immuno-precipitated Tyk2-JH1 kinase were used for kinase assay, in the presence of Brevilin A concentration series at 10, 20, 40, and 80 µM. Western-Blot showed that Brevilin A inhibited Tyk2-JH1 kinase activity directly (A, left). The right lane in (A) was the Coomassie Brilliant Blue staining of purified hSTAT3 after SDS-PAGE. Antibody against pTyr705-STAT3 was used for detecting STAT3 phosphorylation. (B) IC₅₀ measurements of four JAKs-JH1 truncated kinases. HEK293T cells expressing JAKs-JH1 were plated and cultured in 100-mm dishes for 12 h, then media was removed and replaced with fresh media without serum for another 12 h. Cells were then treated with Brevilin A at different concentrations for 4 h (5 µM, 7.5 µM, 10 µM, 12.5 µM, 15 µM, 17.5 µM and 20 µM). DMSO was used as control.