Figure 2.
22-NHC binds Smo at the oxysterol-binding site
(a) Schematic of 22-NHC and 20-OHC affinity matrices. 22-NHC and 20-OHC are covalently attached to agarose beads via a polyethylene glycol (PEG) linker. Control beads carry only the PEG linker.
(b) 22-NHC beads were incubated with detergent extracts of 293T cells expressing mSmo-Cherry, in the presence of the indicated concentrations of competitor compounds. The beads were washed and bound protein was eluted, separated by SDS-PAGE and immunoblotted with anti-Cherry antibodies. A portion of the extract was analyzed in parallel, to show input. MSmo binds specifically to 22-NHC beads, and binding is not competed by other Smo inhibitors.
(c) As in (b) but with the addition of the inactive oxysterol 7-OHC, or the active oxysterols, 20-OHC and 20-OHC-Pent. Binding of mSmo to 22-NHC beads is competed in a dose-dependent manner by 20-OHC and 20-OHC-Pent, but not by 7-OHC.
(d) As in (b) but with the addition of 20(S)-OHC-Pent and 20(R)-OHC-Pent. Binding of mSmo to 22-NHC beads is competed by the active S diastereomer but not by the inactive R diastereomer.
(e) As in (b) but using 20-OHC beads, in the presence of free 22-NHC or 20-OHC. Binding of mSmo to 20-OHC beads is competed in a dose-dependent manner by free 22-NHC and 20-OHC.
The full immunoblots for this figure are shown in Supplementary figure 11.