Xenbase Image ID: 129060

Figure 5. Oxysterol binding to Smo is required for high level Hh signaling(a) Alignment of a portion of CRDs of mouse Smo (mSmo), chicken Smo (gSmo), Xenopus Smo (xSmo), zebrafish Smo (zfSmo), Drosophila Smo (DrSmo) and mouse Fz8 (mFz8). The stretch indicated with red lines contains 5 residues that, in mFz8, contact the Xwnt8 palmityl moiety (Q71, F72, P74, L75 and I78).(b) Cherry-tagged mSmo, mSmoL112D, mSmoW113Y or mSmoS114Y were tested for binding to 22-NHC and 20-OHC beads, in the presence or absence of 20-OHC (100 μM). MSmoL112D and mSmoW113Y do not bind 22-NHC and 20-OHC beads. The full immunoblot is shown in Supplementary figure 11.(c) Smo−/− MEFs rescued with mSmo, mSmoL112D, mSmoW113Y or mSmoS114Y were incubated overnight with DMSO control, SAG (1 μM), 20-OHC (10 μM) or Shh. The graph shows the percentage of Smo-positive cilia. Error bars represent the sub-sampling standard deviation of the fraction of positive cilia (see Online Methods). Between 131–207 cilia were analyzed per condition. MSmoL112D and mSmoW113Y have a defective response to 20-OHC and Shh.(d) As in (c), but with box plots showing Smo fluorescence intensity at cilia. For each condition, the Smo signal was normalized to the intensity of the SAG treatment for the respective cell line.(e) As in (c), but cells were processed for Q-PCR, to measure Gli1 transcription. For each treatment, Gli1 levels were normalized to the level induced by SAG in the respective cell line. Error bars show standard deviation (n=3 independent experiments). MSmoL112D and mSmoW113Y do not respond to 20-OHC and have a reduced responsiveness to Shh. 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

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