Fig. 4. Retention of TSP1 in ECM depends on the C-terminal region in trimeric form. (A) Retention of the indicated TSP1 domain deletion proteins in NH₄OH-insoluble ECM of COS-7 cells detected by indirect immunofluorescence with antibodies to human TSP1 (upper panels) or direct fluorescence of mRFP (lower panels). (B-E) Quantification of the relative expression (B,C) or ECM retention (D,E) of TSP1 domains, as detected by immunofluorescence (B,D) or mRFP (C,E). Each column represents the mean from 3-16 independent experiments, bars represent s.e.m. (F) Biochemical analysis of mRFPovTSP1C from COS-7 ECM. (G) Time course of ECM incorporation of mRFPovTSP1C. mRFPovTSP1C was expressed in COS-7 cells for 18 hours, then cells replated and extracted with 20 mM NH₄OH from 1 hour to 3 days later. Arrows indicate initial deposits of mRFPovTSP1C. The mean cell area at each time point, as measured from ten phalloidin-stained cells in each sample, is given below each panel. Colour panels show X-Y confocal projections of ECM puncta (upper panel; arrow indicates area of merged puncta) or intracellular distribution of mRFPovTSP1C after 3 days (lower panel). (H) Confocal X-Z sections show distribution of mRFPovTSP1C in ECM (upper panel) or paraformaldehyde-fixed cells (lower panel) at 3 days. Scale bars: 10 µm.