Extended Data Fig. 1: Immunoblot validation of bait immunoprecipitations.
From: Outlining cardiac ion channel protein interactors and their signature in the human electrocardiogram
Immunoprecipitated channels Scn5a a), Kcnq1 b), and Cacna1c c) evaluated by western blot. d) Inf2 (Cacna1c interactor) was immunoprecipitated from murine cardiac tissue and evaluated by western blotting for the presence of Cacna1c (left panel, probed with IRDye 800CW secondary antibody) to confirm co-immunoprecipitation with Inf2. The same blot was evaluated for presence of Inf2 (right panel, probed with IRDye 680LT secondary antibody) to ensure that Inf2 was immunoprecipitated. In all the panels, black arrows denote the band of interest. The lowermost arrow in Panel B shows the Kcnq1 monomer band and the higher order oligomers are shown by the upper arrows. UF denotes unbound fraction of the immunoprecipitation (IP) experiment. The immunoblot validation was carried out thrice for panels A-C and twice for panel D with reproducible results. e) LC-MS/MS analysis of Inf2 IPs evaluating Cacna1c (left) and Inf2 protein abundances (right). Triplicate immunoprecipitations were performed from murine cardiac tissue using antibodies against Inf2 or IgG. Precipitated proteins were evaluated by mass spectrometry. Inf2 was abundantly present in the three Inf2 IPs and was absent in the triplicate IgG control Ips (right). The calcium channel protein Cacna1c was identified in all six immunoprecipitations but was a hundred-fold more abundant in the Inf2 IPs than in the control IPs (left).
