Fig. 7. Mutation of the Cardin-Weintraub sequence and disruption of SHH-HSPG interactions does not alter receptor binding. (A) Fixed cultures were incubated with 1 nM of SHH:AP and increasing amount of unconjugated SHH (5-500 nM). Data are the means of triplicate determinations±s.e.m. Linear fits to the steepest region of the bound/free versus bound curve are given beneath each plot along with calculated values for Kd and Bmax. (B) Cells were incubated for 2 hours on ice with increasing doses of SHH:AP or AlaSHH:AP (0.7 to 35 nM) in the absence or presence of 100 nM unconjugated SHH. Specific binding was derived from the difference between triplicate determinations of binding in the absence and presence of excess unconjugated SHH. Specific binding is plotted as net absorbance in OD units at 405 nm. (C) P6 cultures were treated with a mixture of 1 mU/ml heparinase I and III or not as indicated. Specific SHH:AP binding was measured by incubation of 1 nM ligand in the absence (white bars) and presence (black bars) of 100 nM unconjugated SHH. Data are the means of quadruplicate determinations±s.e.m. and are plotted as the fraction of binding measured for SHH:AP alone.
