Xenbase Image ID: 129057

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. Image published in: Nedelcu D et al. (2013) Image downloaded from an Open Access article in PubMed Central. Image reproduced on Xenbase with permission of the publisher and the copyright holder. Larger Image Printer Friendly View

Return to previous page