Lancl2 sirna

Abscisic acid was purchased from PhytoTechnology Laboratories (Overland Park,
KS). LANCL2 siRNA and Control siRNA were obtained from Santa Cruz Biotechnology.
The siRNA vehicle, i-Fect, in the siRNA experiments was obtained from Neuromics
(Edina, MN).

LANCL2 small interfering RNA (LANCL2 siRNA, Santa Cruz Biotechnology, Inc. Ca.)
and scrambled siRNA (Control siRNA, Santa Cruz Biotechnology, Inc. Ca.) were
administered directly into the intrathecal space through lumbar puncture.
Injections were made into the intrathecal space in rats anesthetized with 2%
isoflurane using a 0.5 inch 27 gauge needle connected to a Hamilton syringe as
previously described.^{30 (link),35} LANCL2 siRNA and Control siRNA were prepared
immediately prior to the intrathecal administration by mixing the RNA solution
(100 μM) with transfection reagent (iFect), in a ratio of 1:5 as described in
the iFect siRNA transfection kit.^{36 (link)}
LANCL2 siRNA (2 μg) and an equal amount of Control siRNA in a volume of
10 μl were intrathecally injected at 10:00 a.m. and 10:00 p.m. for two
consecutive days. The hind paw withdrawal response to mechanical stimuli and the
withdrawal response latency to thermal stimuli were measured prior to the
initial lumbar injection and 12 h following the last lumbar puncture. The dorsal
spinal cord at the L4 to L5 region was removed after the behavioral tests for
western blotting.