Figure 2

Intracellular localization of LET-756::GFP and HA-tagged protein partners Cos-1 cells were transfected with let-756::gfp with or without various HA-tagged encoded constructs. Twenty four hours later cells were either fixed in 4% paraformaldehyde and permeabilized in triton or in cold methanol. Cells were incubated for an hour with rat anti-HA antibodies. Secondary goat anti-rat Texas red-coupled antibodies were then added for another 30 min. Coverslips were visualized with a confocal Leica microscope. Cells were transfected with the HA-tagged protein alone (column I) or cotransfected with let-756::gfp and revealed by direct fluorescence (column II), by anti-HA immunofluorescence (column III) and merge (column IV). In cells cotransfected with LET-756 and KIN-10 and treated with actinomycin D, both GFP staining and anti-HA immunostaining are displaced around and into the nucleolus. The yellow star indicates a neighbor single KIN-10 transfected cells. Its localization is not modified by the drugs. Panels A and B each show results for five interactors analyzed.