Fig. 4. Transfection of hNFL clones in SW13 Vim^- cells. (A-D) Transient transfection results of wild-type (A,B), P8R mutant (C) and Q333P mutant (D) hNFL. Staining was carried out with monoclonal anti-NFL antibody. Wild-type hNFL generally assembled into short filaments (A), but occasionally also formed a homopolymeric filamentous network (B) that was not stained with anti-vimentin antibody (not shown). The P8R and the Q333P hNFL mutants (C,D) were not capable of self-assembly into a filamentous network, but formed aggregates. (E-F) Co-transfections and staining of wild-type hNFL with the P8R mutant hNFL (E) and Q333P mutant hNFL (F) showed that the mutant proteins disrupted the self-assembly of wild-type hNFL. Results similar to those in F were obtained with a bicistronic construct that expressed both wild-type and Q333P mutant hNFL (not shown). Bar, 25 µm.