Fig. 5: Detection of sDscam cis interactions on living cell surface.
From: Structural basis for the self-recognition of sDSCAM in Chelicerata
a Schematic diagram of the phosphorylation dimerization assay. The sDscam-cKIT chimera are expressed on Sf9 cell surface. If dimerization occurs, the intracellular portion of cKIT gets phosphorylated, and the phosphorylation signals are then monitored through anti-phospho-tyrosine immunoblotting. b Immunoblotting analysis of cKIT tyrosine phosphorylation. The same samples are detected through anti-phospho-tyrosine and anti-Flag immunoblotting to monitor the phosphorylation protein and the total protein, respectively. Upper panel, anti-phospho-tyrosine immunoblotting. Lower panel, anti-Flag immunoblotting. The Ig7-9 fragment of Drosophila Dscam1 and a nonrelative protein (human NRP1 B1B2 domain) are used as negative controls. For ∆FNIII2 construct, the intermediate FNIII2 domain is replaced by a 15-residue GGS linker. c Verification of the cis-dimer interfaces by mutagenesis study. The FNIII1-FNIII2 interface mutants N357A and Q485A, and the FNIII2-FNIII2 interface mutant Y420A are studied by cKIT tyrosine phosphorylation assay. d, e Quantification of the cKIT tyrosine phosphorylation. The intensities of the bands in b and c are calculated. The phosphorylation signals (anti-phospho-tyrosine immunoblotting intensities) are normalized by the protein level of each sample (anti-Flag immunoblotting intensities). The value of FNIII1-3 is set as 100%. Values are means + SD (n = 3 independent experiments). ***P = 3e-04 (Q485A), ***P = 1e-04 (N357A), ****P < 0.0001, calculated by two-tailed Student’s t test, the exact P values below 1e-04 are not available. Source data are provided as a Source Data file.
