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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Anal Biochem. 2015 Jun 18;488:1–5. doi: 10.1016/j.ab.2015.06.021

Figure 2.

Figure 2

Hh cholesterolysis inhibitor identified by chemical screening. (A) Screening output. Traces of 12 sample wells from a 96 well plate, showing controls, column (1, 12); and compound wells (2-11). Compound in well 7 (green circle) was identified as an potential inhibitor in screens with cholesterol at 60 µM (upper) and 200 µM (lower). (B) Inhibitor structure. TIMTEC chemical identification code ST044643. (C) Effects of ST044643 on C-H-Y. Added ST044643 diminishes FRET from C-H-Y in the pre-incubation period (gray), and limits the extent of reaction compared to the C-H-Y control. (D) Secondary screen of Hh cholesterolysis inhibition by ST044643. Upper: Cholesterolysis activity of chimeric hedgehog precursor, ShhN-DHhC, in the absence and presence of increasing amounts of ST044643. Precursor and products were separated by SDS-PAGE and stained with Coomassie blue. Lower: Relative cholesterolysis activity plotted as a function of increasing ST044643, using data in upper panel. Dose-response curve (solid line) was calculated using an IC50 value of 5 µM.