Western blotting was performed to determine the expression of occludin, alpha-2
spectrin, ERK1/2, phosphorylated ERK1/2 (pERK1/2), AKT, pAKT, and β-actin
(loading control) in the ipsilateral hippocampus of animals in different
experimental groups. The ipsilateral hippocampi were manually homogenized in
ice-cold lysis buffer, and respective supernatants were processed for
electrophoresis as we previously described.34 (link)–36 (link, link),39 (link) Total proteins (25 μg)
from each supernatant were separated by electrophoresis on 4-12%
SDS-polyacrylamide gels (Invitrogen, CA), then transferred to a nitrocellulose
membrane (Sigma, MO). Membranes were blocked with 5% non-fat milk in
Tris-buffered saline with .1% Tween-20 (TBST) and incubated overnight at 4°C in
rabbit polyclonal antibodies against occludin (1:2000; Abcam, MA), α-ii spectrin
(1:3000; Invitrogen/ThermoFisher Scientific, NY) ERK, 1/2, pERK1/2, AKT, pAKT
(1:1000; Cell Signaling Technology, MA) or a mouse anti-β-actin (1:4000; Sigma,
MO). Blots were washed in Tris-buffered saline with .1% Tween-20 and incubated
for 1 h at room temperature with corresponding HRP-conjugated secondary
antibodies (1:5000; Millipore, MA). Horseradish peroxidase-labeled proteins were
detected by enhanced chemiluminescence (ECL, Thermo Scientific, IL), and protein
bands were visualized using a digital blot scanner (LI-COR, NE).
spectrin, ERK1/2, phosphorylated ERK1/2 (pERK1/2), AKT, pAKT, and β-actin
(loading control) in the ipsilateral hippocampus of animals in different
experimental groups. The ipsilateral hippocampi were manually homogenized in
ice-cold lysis buffer, and respective supernatants were processed for
electrophoresis as we previously described.34 (link)–
from each supernatant were separated by electrophoresis on 4-12%
SDS-polyacrylamide gels (Invitrogen, CA), then transferred to a nitrocellulose
membrane (Sigma, MO). Membranes were blocked with 5% non-fat milk in
Tris-buffered saline with .1% Tween-20 (TBST) and incubated overnight at 4°C in
rabbit polyclonal antibodies against occludin (1:2000; Abcam, MA), α-ii spectrin
(1:3000; Invitrogen/ThermoFisher Scientific, NY) ERK, 1/2, pERK1/2, AKT, pAKT
(1:1000; Cell Signaling Technology, MA) or a mouse anti-β-actin (1:4000; Sigma,
MO). Blots were washed in Tris-buffered saline with .1% Tween-20 and incubated
for 1 h at room temperature with corresponding HRP-conjugated secondary
antibodies (1:5000; Millipore, MA). Horseradish peroxidase-labeled proteins were
detected by enhanced chemiluminescence (ECL, Thermo Scientific, IL), and protein
bands were visualized using a digital blot scanner (LI-COR, NE).
