Fig. 5: Functional analyses of the inter-domain interactions in PlexinC1 using the COS-7 cell collapse assay.
a Binding of A39R to PlexinC1 wild type or mutants expressed in COS-7 cells. Cells were treated with His8-tagged A39R at 50 nM for 10 minutes, fixed and subjected to immunostaining with an anti-His-tag antibody. Cells did not undergo collapse after this short period of A39R treatment, therefore suitable for assessing cell surface expression and A39R binding of PlexinC1. A39R and the nucleus are pseudo-colored green and blue, respectively. Δ4, deletion of four residues (938–941) in the linker between the IPT4 and TM of PlexinC1. Ins4, insertion of four residues (GSSG) between residues 939 and 940. Scale bar, 15 µm. One representative image from three biological repeats is shown for each group. b A39R-induced collapse of COS-7 cells expressing PlexinC1 wild type or various mutants. Cells were treated A39R at 50 nM for 90 minutes. FLAG-tagged PlexinC1 was visualized through immunostaining with anti-FLAG antibody and Alexa Fluor 488-labelled anti-mouse IgG secondary antibody. PlexinC1 and the nucleus are pseudo-colored green and blue, respectively. One representative image from three biological repeats is shown for each group. c Quantification of the results from (b). The individual data points, mean and s.e.m of cell collapse percentage from three biological repeats are shown. In total, 96–147 cells in each sample were counted in each of the three biological repeats. Source data are provided as a source data file.
