Fig 1: SK2 interacts with dynein IC. a HEK293 cells were transfected with empty vector (EV) or a vector encoding HA-tagged IC2, either individually or in combination with a vector encoding FLAG-tagged SK2. SK2 was immunoprecipitated from cell lysates with anti-FLAG antibodies, and co-immunoprecipitated IC2 was detected by immunoblotting with anti-HA antibodies. Expression levels of IC2 in the lysates were confirmed by immunoblotting with anti-HA antibodies (Lysate). Immunoprecipitates were also probed with anti-FLAG antibodies to confirm the presence of SK2. Blots shown are representative of at least five independent experiments. b HEK293 cells were transfected with empty vector (EV) or a vector encoding FLAG-tagged SK2, either individually or in combination with a vector encoding HA-tagged IC1 or HA-tagged IC2. Lysates were pre-cleared with Protein G µbeads. Co-immunoprecipitation and immunoblotting analyses were then performed as described in (a). Blots shown are representative of three independent experiments. c SK2 was immunoprecipitated from murine whole brain lysate using anti-SK2 antibodies. Co-immunoprecipitated dynein intermediate chains (IC), light intermediate chain 1 (LIC1) and dynactin p150 were detected by immunoblotting with anti-IC, anti-LIC1 and anti-dynactin p150 antibodies, respectively. Expression levels of these proteins in the mouse brain lysate were confirmed by immunoblot analyses with their respective antibodies (Lysate). Lysates and immunoprecipitates were also probed with anti-SK2 antibodies to confirm expression and immunoprecipitation of SK2. H/C designates the heavy chain IgG band. Blots shown are representative of three independent experiments. d Immunofluorescence staining and confocal microscopy demonstrating co-localization of SK2 and dynein IC in HEK293 cells. SK2 (green) was detected using anti-SK2 antibodies and dynein IC (red) was detected using anti-IC antibodies. Nuclei were highlighted using DAPI (blue). Images are representative of at least 100 cells, from three independent experiments. Scale bar = 10 μm. e Immunofluorescence analysis and confocal microscopy demonstrating direct interactions between SK2 and dynein IC, using the Duolink® in situ PLA system with anti-SK2 (1:300; ECM Biosciences) and anti-IC antibodies (1:300) in HEK293 cells (top panels). Each red dot indicates a single direct interaction. Nuclei were highlighted using DAPI (blue). Differential interference contrast images are also shown (bottom panels). Images are representative of at least 100 cells, from three independent experiments. Scale bar = 10 μm