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. 2019 Nov 19;9:17104. doi: 10.1038/s41598-019-53176-4

Figure 4.

Figure 4

Signalling-defective DDR1 mutants bind triple-helical DDR1 selective peptide but do not phosphorylate with peptide stimulation. (A) COS-7 cells transiently expressing wild-type DDR1 or the indicated DDR1 mutant were incubated with or without a biotinylated DDR-selective collagen-mimetic peptide for 60 minutes on ice, followed by incubation with anti-DDR1 mAb 7A9 on ice. Cells were then fixed and incubated with Alexa Fluor-488 goat-anti-mouse IgG and Alexa Fluor-546 conjugated streptavidin. Cells were imaged by widefield microscopy. The graph shows mean fluorescence intensity, normalised to respective DDR1 expression levels. N = 27–31 fields of view from 3 independent experiments. Scale bar, 20 μm. (B) HEK293 transiently expressing wild-type DDR1 or the indicated DDR1 mutant were stimulated with collagen I (C), or with DDR-selective collagen-mimetic peptide (P) for 60 minutes at 37 °C or were left unstimulated. Cell lysates were analysed by Western blot using an Ab against phosphorylated Tyr-513 (anti-pY). The blot was stripped and re-probed with anti-DDR1. The positions of molecular mass markers are indicated on the left in kDa. The bar chart shows the densitometry analysis of pY513 band intensities after normalization to total DDR1. Each value is a percentage of the sum of all the pY513/DDR1 signals on the blot. The graph shows mean band intensities + SEM (N = 3). NS, no significance; *p < 0.05; **P < 0.01; ****p < 0.0001 (two-way ANOVA, followed by Tukey’s multiple comparisons test. (C) COS-7 cells transiently expressing DDR1 were incubated with collagen-mimetic triple-helical peptide (Peptide) or a control triple-helical peptide without the DDR binding motif (Control) for 0 to 60 minutes at 37 °C, as indicated. Cells were then incubated with anti-DDR1 mAb7A9 Ab on ice, before fixation and incubation with secondary Abs. Lower panels show magnified views of the boxed areas in the upper panels. Cells were imaged by widefield microscopy. Scale bars, 20 μm (upper image) or 10 μm (magnification). Right: ZC score of surface DDR1 staining in cells stimulated with Control or Peptide for 0 to 60 minutes. Data show mean + SEM (N = 200–300 regions from 35–50 cells from 2 independent experiments). NS, no significance; ***p < 0.001 (one-way ANOVA, followed by Bonferroni post hoc test).