Fig. 3. Wild-type and mutant MAP4 colocalize with MTs in mouse L^tk- cells. (A) GFP fluorescence and anti-tubulin-stained images of L^tk- cells stably expressing WT-, KK-, AA-, or EE-MAP4 mutants. Cells were induced with dex for 36 hours, fixed in methanol, and stained with 3F3, a monoclonal tubulin antibody. Note that, because fixation decreases the brightness of GFP fluorescence, the micrographs shown accentuate images of bright, clustered or bundled GFP-EE-MAP4 MTs, compared with single MTs. By contrast, in live cell imaging, GFP fluorescence of single MTs was easily detected (data not shown), confirming that the MT distribution in GFP-MAP4 cells was indistinguishable from that of L^tk- cells. (B) Immunoblots of L^tk- cell extracts containing transfected MAP4 constructs. Two clones each of transfectants expressing wild-type (WT) and mutant MAP4 form were isolated, and 50 µg of each cell extract was immunoblotted with an antibody against human MAP4 (top lanes) and 3F3 tubulin antibody (bottom lanes). Live GFP fluorescence of EE-MAP4 from the last clone was visible under microscopy at low levels, but EE-MAP4 was not readily detected by western blotting. This clone of cells showed a very low level of expression (see Table 1 for details).