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Pull-down and MS analyses expand the CEP76 interactome. (A) Graphical representation of seminative co-IP assay. mCherry-tagged DNA plasmids (WT CEP76 or p.Val24Asp CEP76 or empty mCherry vector) were independently overexpressed in U2OS cells, harvested in IP lysis buffer at 60 hpt and immunoprecipitated with an anti-mCherry antibody. The interaction partners were detected by MS analysis and validated by antigen-specific immunoblotting (n = 3 biological replicates). (B) Molecular interactions of CEP76. Interaction partners identified in MS analysis were plotted in Cytoscape (https://cytoscape.org/), and known and predicted protein-protein interactions from String were integrated into the experimental data to generate the CEP76 interactome. Proteins identified in the CEP76 precipitate that were part of an established network are shown on the right, and candidate CEP76-interacting proteins are on the left inside a dotted red circle. (C) Western blots of coimmunoprecipitated proteins. One-fifth of the IP eluate was migrated on 4 to 15% polyacrylamide gels, and proteins of interest (CEP76, CCP110, LUZP1, and mCherry) were detected by protein-specific antibodies (n = 3 biological replicates).
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