Figure 2.

Coumermycin-induced changes in PPME binding activity by L-Gb–expressing 300.19 cells. (A) Immunofluorescence analysis of PPME binding by cells expressing wild-type L-selectin or L-Gb, before and after treatment with 0.9 μM coumermycin. After coumermycin treatment, the cells were incubated with PPME in the presence of either Ca²⁺ or EDTA, and PPME binding was assessed by fluorescence staining and flow cytometry analysis with results shown on a three-decade log scale. These results represent those obtained in at least five experiments with the L-Gb clone shown (Fig. 1 B) and are representative of results obtained with two independent clones of L-Gb–transfected cells. (B) Dose–response of coumermycin-induced PPME binding in L-Gb cells. Values represent the mean fold increase in PPME binding relative to untreated cells obtained in four experiments. Asterisk indicates significant differences between treated and untreated samples, P < 0.01, Student's t test. (C) Time kinetics of coumermycin-induced PPME binding by L-Gb transfectants. The cells were treated with 0.9 μM coumermycin for the indicated amounts of time before PPME staining. Asterisk indicates significant differences between treated and untreated samples, P < 0.05. (D) Inhibition of coumermycin-induced PPME binding by novobiocin. Cells were first treated with medium or the indicated amounts of novobiocin at 37°C for 15 min. Coumermycin (0.9 μM final) was then added with PPME binding assessed 25 min later. (E) Effect of coumermycin treatment on L-selectin expression. L-Gb cells were incubated in media containing DMSO (0.1%), 0.9 or 9.0 μM coumermycin at 37°C for the indicated time periods. The cells were washed and L-selectin expression was assessed as in Fig. 1. Values represent mean ± SEM fluorescence channel numbers obtained in three experiments.