Rabbit anti akt

BD Matrigel™ Basement Membrane Matrix, culture dishes, and plates were obtained from Corning Inc. (Corning, NY, USA). Concanavalin A (Con A), sodium dodecyl sulphate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Endothelial Cell Medium (ECM) was obtained from ScienCell (Carlsbad, CA, USA). PD98059 and LY294002 were the products of Tocris Bioscience (Bristol, UK). The primary antibodies, such as rabbit anti-phospho-Akt (Ser473) (#4060S), rabbit anti-Akt (#9272S), rabbit anti-phospho-ERK1/2 (#4730S), rabbit anti-ERK1/2 (#4695S), rabbit anti-phospho-p38 (#4511S), rabbit anti-p38 (#8690S), mouse anti-p27 (#3686S), mouse anti-p21 (#2947S), rabbit anti-cyclin D1 (#55506S), and rabbit anti-cyclin E (#20808S) antibodies were from Cell Signalling Technology (Beverly, MA, USA). The polyvinylidene fluoride (PVDF) membranes were the products of Millipore (Billerica, MA, USA). EdU Apollo®488 In Vitro Imaging Kit was a product of RIBOBIO (Guangzhou, China).

At 3 days after surgery, mouse hippocampal Cornu Ammonis 3 (CA3)/DG regions were collected, and lysed with ice‐cold homogenization buffer containing 2% protease inhibitor cocktail (Thermo Scientific). The samples containing equal amounts of protein were separated on 10% sodium dodecyl sulfate‐polyacrylamide gels (SDS‐PAGE), and transferred to PVDF membranes. After blocking with 5% nonfat milk and 0.1% Tween 20 in Tris‐buffered saline (TBS) at room temperature for 2 h, the membranes were incubated at 4℃ overnight with rabbit anti‐pERK1/2 (1:2500, Cell Signaling), rabbit anti‐ERK1/2 (1:2000, Cell Signaling), rabbit anti‐pAkt (1:2000, Cell Signaling), rabbit anti‐Akt (1:1000, Cell Signaling), and mouse anti‐β‐actin (1:10,000, Sigma). After washing, the membranes were incubated with peroxidase‐conjugated goat anti‐rabbit IgG (1:10,000, Sigma) or goat anti‐mouse IgG (1:10,000, Sigma) for 1 h at room temperature. The target protein signal was detected using a chemiluminescence detection kit (Pierce™ ECL western blotting Substrate, Thermo Scientific) and visualized on an electrophoresis image analyzer (Tianneng). β‐Actin was employed as the loading control. The band intensity was analyzed using Image J software. Quantification of densitometric values was calculated as follow: [(phospho‐protein/β‐actin)]/[total‐protein/β‐actin)] and expressed as ratio relative to the sham group.