Fig. 1. Sequence alignment of NFL clones, location of mutations and western-blotting of transfected proteins with anti-NFL antibody. (A) Multiple sequence alignment of the hNFL protein encoded by our hNFL cDNA (hNFL), the previously published hNFL sequence (hNFL P09716), and the published rNFL sequence (rNFL). All the differences in the protein sequences are marked by vertical boxes. Differences in the amino acid sequence between hNFL and hNFL (P09716) are marked with an asterisk (^*), while amino acids that differ between hNFL and both hNFL (P09716) and rNFL are indicated with a hash mark (#). (B) Schematic representation of NFL protein and the positions of the mutant and variant amino acids. The head, rod and tail domains are indicated. The rod domain is divided into coils 1A, 1B, 2A and 2B separated by linkers L1, L12 and L2. The CMT2 mutations are: P8R in the head domain and Q333P in the coil 2B. The D469N variant is in the tail domain. (C) Western blots of transfected human (left) and rat (right) NFL proteins. Cytoskeletal extracts of transfected cells were separated by SDS-PAGE and subjected to western blotting with a polyclonal anti-NFL antibody. The antibody recognized a band of the same size in wild-type NFL (wt), variant NFL (D469N), head mutant NFL (P8R), and rod mutant NFL (Q333P). The bar indicates the position of the 64 kDa size marker.