P akt and total akt

Animals were sacrificed after 4 days of binge ethanol exposure. Brains were harvested on ice and homogenized in RIPA lysis buffer (Beyotime, Shanghai, China). Each sample was a mixture of hippocampal and prefrontal cortex tissue from at least four rat brains. Brain tissue protein extracts were separated by SDS‐PAGE, transferred to nitrocellulose membranes (Millipore, Billerica, MA) and probed with the following primary antibodies: α‐tubulin (1:1000), β‐Actin (1:1.000), PI3K (1:200) (Santa Cruz Biotechnology, CA); p‐ERK and total ERK, p‐Akt and total Akt, p‐TrkA and TrkA, p‐TrkB and TrkB (1:1000, Cell Signaling Technology, Danvers, MA); doublecortin antibody 3E1 (DCX), tubulin‐β‐III (Tuj‐1,1:1000, Novus Biologicals, St. Charles, MO), microtubule‐associated protein 2 (MAP2, 1:1000, Synaptic Systems, Gottingen), and BDNF (1:500, Abcam). Blots were then probed with appropriate HRP‐conjugated secondary antibodies (Santa Cruz Biotechnology, CA), and immunoreactive proteins were detected using the ECL detection system (Thermo, Rockford, IL). Western blot analysis was performed on samples from three separate experiments and each sample was a mixture of tissues from four rat brains per group. Band optical densities were quantified with Image J software (NIH, MD).