Figure 2

Collagen I induces DDR1 redistribution on the cell surface. COS-7 cells transiently expressing DDR1 were stimulated with collagen as detailed below. (A) Cells were stimulated for the indicated times (in minutes) at 37 °C, then incubated on ice with mAb 7A9 against the DDR1 ectodomain, before fixation and secondary Ab staining. (B) Cells were stimulated for the indicated times at 37 °C. Staining was done as above. The graph shows mean ZC scores + SEM (N = 200–400 regions from 80–100 cells from 3 independent experiments). *p < 0.05; ****p < 0.0001 (one-way ANOVA, followed by Bonferroni post hoc test). Data are from a different set of experiments than those shown in panel A. (C,D) Cells were stimulated for the indicated times at 37 °C, then fixed and permeabilised, and immunostained for phospho-tyrosine 513 (pY-DDR1) and for DDR1. (C) Mean pY-DDR1 levels across three experiments: mean values were normalised within experiments and then mean values taken for each stimulation time. N is at least 30 for each stimulation time. (D) The proportion of cells expressing DDR1 with pY-DDR1 signal above background levels were manually counted for different stimulation times. Mean percentage values ± SEM (N = 55 for all stimulation times, from two independent experiments). (E) Cells were either stimulated with collagen I for 10 minutes at 37 °C or left unstimulated, then incubated on ice with mAb 7A9 against the DDR1 ectodomain (shown in green) and anti-collagen-I mAb (shown in magenta), before fixation, and secondary Ab staining. White boxes in left columns indicate corresponding areas shown at higher magnification in columns to the right. All cells were imaged using a widefield microscope. At least 30 cells were imaged for each condition. Scale bars, 30 μm or 10 μm (enlarged images).