Semaphorin III binding to MS-5 cells. Sub-confluent MS-5 cells were treated for 90 minutes with different concentrations of semaIII-AP. Then, AP activity from bound semaIII-AP was measured colorimetrically (see Materials and Methods for details). Specific binding was determined by substraction of values obtained from binding to MS-5 cells and to COS cells. COS cells do not express neuropilin-1 and are unable to bind semaIII-AP.8 (A) Scatchard’s analysis. The values shown are an average of 3 different assays. Linear regression analysis of values showed that MS-5 cells express 2,500 binding sites per cell and bind Semaphorin III with a kd of 1.2 × 10⁻¹⁰ mol/L. (B) MS-5 cells were incubated with concentrated conditioned medium containing 5 ng/mL of semaIII-AP in the absence or in the presence of indicated concentrations of VEGF 165. Values shown are an average of 2 different assays. Reaction time for AP activity detection was 3 hours.