Fig. 2. Characterization of the subcellular distribution of eIF3k. (A) eIF3k colocalizes with K8 and K18. HeLa and HaCaT epithelial cells were fixed, double stained with anti-eIF3k antiserum and antibody to K8 or K18, and examined by epifluorescence or confocal microscopy as indicated. One confocal section corresponding to each individual labeling is shown. The overlay of green and red images is shown in the `Merge' panel. (B) eIF3k does not colocalize with F-actin or microtubules. HeLa cells were fixed, double stained with eIF3k antiserum and either rhodamine-phalloidin or anti--tubulin, and examined by confocal microscopy. (C) Immunofluorescence analysis of eIF3k distribution in cells without keratin filaments. SW13 and NIH3T3 cells were immunostained with the eIF3k antiserum and then examined by epifluorescence microscopy. (D) The eIF3k antiserum does not crossreact with an epitope in K18. The eIF3k antiserum was pre-absorbed with 100 µg recombinant K18. HeLa cells were double stained by the pre-absorbed eIF3k antiserum (red) and anti-K18 antibody (green), and were examined by epifluorescence microscopy. (E) HeLa cells stably expressing eIF3k siRNA and GFP (see Fig. 5A for description) were mixed with parental HeLa cells and then examined by immunostaining with eIF3k antiserum. (F) Distribution of eIF3k in both the detergent-soluble and -insoluble compartments of cells. HeLa cells were extracted with lysis buffer containing 0.5 % Triton X-100 (see Materials and Methods). The detergent-soluble and -insoluble fractions were collected and one fifteenth of each fraction was separated by SDS-PAGE followed by western blot analyses with antibodies as indicated. CrkII and K8 were used as the reference protein for soluble and insoluble fractions, respectively. The percentages indicate the amount of eIF3k in each fraction. Scale bars: 5 µm.