Proteins were analyzed by SDS–polyacrylamide gelelectrophoresis. The
concentration of acrylamide of the resolving gel varied from 7.5 to 15%. The
resolved proteins were transferred to nitrocellulose membranes (Amersham
Hybond-ECL, GE Healthcare, Buckinghamshire, UK) in Tris-glycine buffer
containing 20% methanol. The membranes were first dried, washed with
Tris-buffered saline (10 mM Tris, pH 7.5,
150 mM NaCl) and then blocked, or directly blocked for
1 h in Tris-buffered saline containing 5% bovine serum albumin. The
blots were incubated with the primary antibodies in PBS containing 0.05% Tween
20, for 12 h at 4 ºC. After washing with
Tris-buffered saline containing 0.05% Tween 20, the membranes were incubated
with Odyssey IRdye CW 800 secondary antibodies (LI-COR Biosciences) for
1 h at room temperature. After washing, the blots were imaged using
an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
concentration of acrylamide of the resolving gel varied from 7.5 to 15%. The
resolved proteins were transferred to nitrocellulose membranes (Amersham
Hybond-ECL, GE Healthcare, Buckinghamshire, UK) in Tris-glycine buffer
containing 20% methanol. The membranes were first dried, washed with
Tris-buffered saline (10 m
150 m
1 h in Tris-buffered saline containing 5% bovine serum albumin. The
blots were incubated with the primary antibodies in PBS containing 0.05% Tween
20, for 12 h at 4 ºC. After washing with
Tris-buffered saline containing 0.05% Tween 20, the membranes were incubated
with Odyssey IRdye CW 800 secondary antibodies (LI-COR Biosciences) for
1 h at room temperature. After washing, the blots were imaged using
an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).