HGF recruits additional synapsin 1 to MET receptor complex.

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HGF recruits additional synapsin 1 to MET receptor complex. HGF recruits additional synapsin 1 to MET receptor complex. A, Quantitative analysis of PLA signals generated with β-catenin alone (β-cat), β-catenin with MET (+ MET), β-catenin with synapsin 1 (+ Syn 1), β-catenin with Neurexin 1 (+ Nxn 1), and β-catenin with PSD-95 (+ PSD-95). Error bars represent the SEM; N = 6–8 cells from independent cultures for each group. B, Representative Western blots of complexes immunoprecipitated from P14 cortical crude synaptosomes using an anti-MET or control IgG antibody. This experiment was repeated three times using independent synaptosomal preparations. In PBS-treated synaptomsomes, synapsin 1 and MET are detected in the MET, but not IgG, pulldowns. Stimulation of the synaptosomes for 5 min with HGF increased the MET/synapsin 1 complex (HGF vs PBS lane). An anti-phospo-MET antibody (α-pMET) was used to confirm HGF-induced activation of the MET receptor. C, Representative confocal microscopy images of PLA staining in primary cultures of neocortical neurons at 14 DIV following treatment with PBS or HGF for 10 min. Red fluorescent profiles represent regions of PLA signal amplification denoting MET and synapsin 1 colocalization. For comparison, the total MET immunoreactivity (green fluorescence) in the same field is illustrated. Scale bar, C (for B, C), 5 μm. D, Quantitative analysis of the MET/synapsin 1 PLA signals. Error bars represent the SEM; N = 30 cells from five independent cultures in each group. *p < 0.05 (HGF vs PBS). E, Representative Western blots of complexes that were immunoprecipitated from P14 cortical crude synaptosomes using an anti-MET or control IgG antibody. MET is detected in the MET, but not IgG, pulldowns. Stimulation of the synaptosomes for 5 min with HGF results in phospho-MET detection in MET pulldowns (HGF vs PBS lane). A single synaptophysin 1 band is readily detected in the input sample prior to IP. In contrast, the postimmunoprecipitation sample has only nonspecific bands in all Co-IP groups, indicating that synaptophysin 1 does not coimmunoprecipitate with MET under these conditions. Zhihui Xie et al. eneuro 2016;3:ENEURO.0074-16.2016 ©2016 by Society for Neuroscience