Fig. 5. SHH binds to endogenous HSPGs through the Cardin-Weintraub sequence. The binding of SHH:AP and AlaSHH:AP to endogenous HSPGs in postnatal day 3 and 6 cerebellum was evaluated by modification of the method of Friedl (Friedl et al., 1997). (A) SHH:AP and AlaSHH:AP exhibit very low levels of binding (red) to sections from P3 mice. DAPI-stained nuclei appear blue. Significant amounts of SHH:AP but not AlaSHH:AP are bound by P6 HSPGs, suggesting that SHH:AP requires a wild-type Cardin-Weintraub sequence for this binding. Scale bar: 10 µm. (B) Heparinase I and III treatment results in significantly decreased binding of SHH:AP to the EGL and IGL in sections of P6 cerebellum. Scale bar: 10 µm. (C) Higher magnification view of the SHH:AP localization. Mutation of the Cardin-Weintraub sequence (AlaSHH:AP) reduces the total level of binding. Scale bar: 10 µm.
