Figure 3

Collagen binding and collagen-induced DDR1 redistribution of signalling defective DDR1 mutants. COS-7 cells transiently expressing WT-DDR1 or the indicated DDR1 mutant were stimulated with collagen I as detailed below. (A) Cells were stimulated with collagen for 10 minutes at 37 °C or left unstimulated, then incubated on ice with mAb 7A9, before fixation and secondary Ab staining. White boxes in left columns indicate corresponding areas shown at higher magnification in right columns. (B) Cells were stimulated with collagen for 60 minutes at 37 °C or left unstimulated, then incubated on ice with mAb 7A9 (shown in green) and anti-collagen I mAb (shown in magenta), before fixation, and secondary Ab staining. Graph shows the quantified anti-collagen signal with the exclusion of collagen not colocalised with DDR1. The mean collagen-immunostain intensity was calculated for each condition then normalised so that WT-DDR1 collagen-unstimulated and stimulated values were 0 and 100 A.U., respectively. Error bars are SEM (N = 65–121 cells from 3 independent experiments). (C) Cells expressing WT-DDR1 were incubated with either collagen I, a mixture of collagen I and anti-DDR1 mAb 7A9, or left unstimulated for 60 minutes at 37 °C. Cells were then immunostained as in B. Arrows indicate collagen aggregates not colocalising with DDR1. Cells were imaged using a widefield microscope. Scale bars, 30 μm or 5 μm (enlarged images in A). For each condition, at least 20 cells were imaged in A, and at least 30 cells were imaged in C.