Anenhanced chemiluminescence detection kit

Whole-cell lysates were prepared by incubating cell pellets in lysis buffer [30
mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4), 1 mM
Na₃VO₄, 25 mM NaF, 10 mM
Na₄P₂O₇] for 30 min on ice. After the
insoluble fractions were removed by centrifugation at 13,000×g at
4°C for 30 min, the supernatants were collected and protein concentration
was determined with a BCA protein assay kit (Pierce Biotechnology, Woburn, MA,
USA). The same amounts of proteins (~30 μg) were subjected to
SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were
incubated for 1 h at room temperature (RT) with a primary antibody in
Tris-buffered saline containing 0.05% Tween-20 [TBS-T (pH 7.4)] in the presence
of 5% nonfat dry milk. After the membranes were washed in TBS-T, secondary
antibody reactions were performed with an appropriate source of antibody
conjugated with horseradish peroxidase. The signals were detected with an
enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech) in the
LAS-3000 detector (Fujifilm, Tokyo, Japan). Immunoblotting for β-actin
was performed in every experiment as an internal control.