coli expression system in order to obtain a sufficient amount of GST-fused m3MST (GST-3MST) for the HTS (about 30 mg of GST-3MST from 900 mL of LB medium; Supplementary Fig. S1). Figure 1: HTS scheme and hit compounds. (a) The fluorescent probe for H2S, HSip-1, and its ...
in the coupled sequential kinase reactions with MST2 or MAP4K4, which was immunopurified from transfected HEK293A cells in a mild lysis buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 50 mM NaF, 2 mM EDTA, 1% NP40 substitute). 30 min after kinase reaction, recombinant GST-...
(d) A scheme of the in vitro human kinome screening for LATS1/2-HM kinases. Flag-tagged individual kinases were expressed and purified from HEK293A cells by anti-flag immunoprecipitation under high stringency, and then applied to the in vitro kinase assays with the truncated form of ...