Fig. 6. Transient transfection of hNFL constructs in SW13 Vim⁺ cells. Transfection of wild-type (A,B), P8R mutant (C,D) and Q333P mutant (E-H) hNFL. The cells were double-labeled with monoclonal anti-vimentin (A,C,E,G) and polyclonal anti-NFL (B,D,F,H) antibodies. Both wild-type hNFL (A,B) and D469 hNFL (not shown) incorporated into the endogenous vimentin network. The P8R mutant hNFL protein incorporated into the endogenous vimentin network in most cases (C,D). The co-assembly of the Q333P mutant hNFL with endogenous vimentin was dependent on their relative levels of expression (E-H). At lower levels of expression the mutant Q333P hNFL incorporated into the vimentin network (E,F), while at higher levels of expression it aggregated in the cytoplasm (G,H). Bar, 25 µm.