Fig. 2
SEC5 interacts with InsP3R. a Interaction between SEC5 and InsP3R3 in RAW264.7 cell lysates. The cell lysates were immunoprecipitated (IP) with control rabbit IgG, anti-SEC5, or anti-InsP3R3 antibody. The lysate (Input) and IP samples were analyzed by western blotting with the indicated antibodies. b Confocal images showing SEC5 and InsP3R co-localization in BMDM cells (Pearson coefficient = 0.71). A representative resting BMDM cell was immunostained with anti-SEC5 (red) and anti-InsP3R3 antibodies (green). c Representative images of FRET donor (Alexa Fluor 488) and acceptor (Cy3). FRET pair intensities before and after Cy3 was photobleached are shown (left and middle columns). The histogram summarizes FRET efficiency in multiple regions of interest (data are summarized as the mean ± SEM; n = 16 for each pair of samples; ***p < 0.001). d Schematic illustration of the functional domains of rat InsP3R type 1 and GST fusion proteins (H1 to H4) of the InsP3R C-terminus (aa 2573–2749) used in SEC5 pull-down assays. A representative western blot depicting the GST pull-downs of SEC5 from RAW267.4 cell extracts is shown. The Coomassie blue-stained gel shows input GST-tagged H1 to H4 fragments. e Schematic diagram of recombinant SEC5 fragments used for testing interactions with InsP3R H1 helices (upper panel). Representative in vitro pull-down assays depict the interaction of different His-tagged SEC5 fragments with GST-InsP3R-H1