Immunofluorescence and Western Blot Protocols

Both immunofluorescence assays (IFAs) and Western blots were performed with previously described protocols (22 (link)). For this study, the primary antibodies used were rabbit anti-HA (Cell Signaling Technology) and rat anti-IMC3, both used at 1:1,000. For the IFA, the secondary antibodies were Alexa Fluor 594- or Alexa Fluor 488-conjugated goat anti-rabbit and goat anti-rat (Invitrogen), both used at 1:2,000. Imaging was performed with a Nikon Eclipse E100080i microscope at ×100 magnification. For Western blots, we used anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP)-labeled IgG.