1. Interaction of NF-L with C1 cassette-containing NR1 splice variants.
Interaction of NF-L with C1 cassette-containing NR1 splice variants. A, B, Ten micrograms of purified fusion protein corresponding to NR1a-Cterm or NR1c-Cterm were dissolved in 8 m urea in the presence or absence of 100 μg of purified NF-L and dialyzed against a buffer containing 100 mm MES, pH 6.5, 170 mm NaCl, 1.0 mmDTT, and 0.5 mm EGTA (see Materials and Methods). Polymerized neurofilaments were then sedimented by centrifugation at 150,000 × g. Proteins present in the supernatants (S) and pellets (P) were resolved by SDS-PAGE and visualized by Coomassie blue staining. Ten micrograms each of NR1a-Cterm and NR1c-Cterm were run as controls (left two lanes). The arrowhead indicates NF-L, the solid arrow indicates NR1a-Cterm, and thedashed arrow indicates NR1c-Cterm. C, QT6 cells expressing either NR1a or NR1c were solubilized, and the cell lysates were incubated with either 6xHisBB or NFL-1 6xHis. Bound protein complexes were precipitated using Ni+-agarose, resolved by SDS-PAGE, and subjected to immunoblot analysis using an anti-NR1 antibody. Thearrowhead indicates precipitated NR1 protein. InB and C, molecular mass markers in kilodaltons are shown.
Michael D. Ehlers et al. J. Neurosci. 1998;18:
©1998 by Society for Neuroscience