Figure 4.

Assemble of rSlo and rANKRA in transfected cells. (A) rSlo can be coimmunoprecipitated with HA-tagged rANKRA protein. Protein complexes were immunoprecipitated with rabbit polyclonal α-HA antibody from lysates of CHO-K1 cells transfected with rslo/pcDNA3.1 and pTracer-CMV, rslo/pcDNA3.1, and HA-tagged rANKRA/pTracer-CMV, or pcDNA3.1 and HA-tagged rANKRA/pTracer-CMV. Precipitated proteins were separated in 6% SDS-PAGE and probed with anti-hSlo antibody through immunoblotting. Slo-immunoreactive protein (∼130 kDa) was detected in the HA-immunoprecipitants. HC, IgG heavy chain. Relative positions of molecular standard are indicated (kilodaltons). (B) Proteins were reciprocally precipitated with mouse monoclonal α-hSlo antibody and probed with rabbit polyclonal HA antibody. Samples were separated in 12% SDS-PAGE. Immunoprecipitation procedures were validated by control experiments for detecting and precipitated rSlo protein and IgG light chain (LC). (C–E) Immunofluorescence microscopy of transiently transfected COS-7 cells by using rSlo::EGFP and HA-rANKRA proteins is shown. rSlo::EGFP channels were directly monitored under fluorescence microscope (Ci, green), and HA-rANKRA proteins were stained with Texas Red-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) following rabbit anti-HA antibody (Di, red). The merged image was constructed using software Adobe Photoshop. Merged signals due to the colocalization of proteins are detected (white arrows) along the cell surface (Ei, yellow). Boxed regions marked in Ei were magnified for better visualization of colocalization (Cii, Dii, and Eii). Bar (C–E), 20 μm.