Synthesis of NLS Peptide

High-purity (>95%) S413-PV peptide: ALWKTLLKKVLKAPKKKRKVC)

reverse NLS peptide (ALWKTLLKKVLKAVKRKKKPC)

and scrambled sequence (KTLK-VAKWLKKAKPLRKLVKC)

peptides were modified with an acetyl group at the N-terminus
and had an amide group at the C-terminus. Peptides were tagged
with Nanogold (Monomaleimido Nanogold, Nanoprobes, NY,
d 1.4 nm) by forming a covalent bond between the thiol group
of peptide and the maleimide group of label by incubation at a
molar ratio 2.5:1 in 50% methanol at 30 °C for 90 min.
Methanol was removed and the conjugate concentrated by
speed-vac to reach 50-100 ÎŒM concentration of labeled peptide.
The conjugate with 1:1 peptide/Nanogold ratio was purified to
homogeneity by size-exclusion chromatography on Superdex
Peptide HR10/30 in buffer containing 35 mM Tris-HCl pH 7.5,
105 mM NaCl, and 30% (v/v) acetonitrile or by reversed phase
chromatography on Pro RPC HR5/2 (Pharmacia, Sweden) in
water-acetonitrile gradient in the presence of 0.1% TFA and
concentrated again.

Treatment of Cells with Peptides. Cells were seeded onto
glass coverslips in 24-well plates, grown to 80-90% confluence,
and incubated with culture medium containing 0.1-3 ÎŒM gold-
labeled peptides at 37 °C for 10 min to 24 h depending on the
experimental setup. After incubation, the cells were washed
twice with cell culture medium and processed for electron
microscopy (see below). For low-temperature experiments, cells
were preincubated in culture medium at 10 °C for 30 min,
followed by treatment with peptides at the same temperature
for 1 h.