Fig. 5

Multiple myosins co-immunoprecipitate recombinant Na⁺/K⁺-ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na⁺/K⁺-ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na⁺/K⁺ ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies (a and b; lanes 2 and 5) or mouse IgG1 antibodies (c; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes (a; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes (b; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes (c; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na⁺/K⁺-ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na⁺/K⁺-ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na⁺/K⁺-ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12: Figure S11, Additional file 13: Figure S12, Additional file 14: Figure S13)