Rabbit anti akt

PLTMax^® Human Platelet Lysate was purchased from Merck (Darmstadt, Germany). Fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA). PD98059 (an inhibitor of MEK), LY294002 (an inhibitor of phosphatidylinositol-3-kinase-AKT), and SB203580 (an inhibitor of p38) were all from Calbiochem-Novabiochem (San Diego, CA, USA). SP600125 (an inhibitor of JNK) was from Sigma (St. Louis, MO, USA). Rabbit anti-phospho-ERK1/2 was from Epitomics Inc. (Burlingame, CA, USA). Rabbit anti-phospho-AKT and rabbit anti-AKT were from Abcam (Cambridge, UK). Rabbit anti-ERK1/2 and rabbit anti-β-actin were from Cell Signaling Technology (Beverly, MA, USA). Heparin sodium injection-N was purchased from Mochida Pharmaceutical CO., LTD. (Tokyo, Japan). Antibodies of CD90, CD31, CD45, and CD34 were purchased from Beckton Dickinson Pharmingen (San Diego, CA, USA). All the other reagents, unless specified otherwise, were purchased from Sigma (St. Louis, MO, USA).

Total proteins from the hippocampus were homogenized on ice in 100 μl lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) plus protease inhibitors. The extract was centrifuged, and the supernatant was collected. Protein concentrations were measured by the BCA Protein Assay Kit (Beyotime Biotechnology, China). Equivalent amounts of protein were loaded in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with TBST containing 5% skim milk for 2 h at room temperature. Then, the membranes were treated overnight at 4°C with the different antibodies: β‐actin (1:2000; Cell Signaling), rabbit anti‐BDNF (1:500; Abcam), rabbit anti‐ERK1/2 (1:1000; Cell Signaling), rabbit anti‐pERK1/2 (1:500; Cell Signaling), rabbit anti‐AKT (1:1000; Abcam), rabbit anti‐pAKT (1:500; Abcam), rabbit anti‐CREB (1:1000; Cell Signaling), and rabbit anti‐pCREB (1:500; Cell signaling). Next, the membranes were incubated with the respective secondary antibodies for 2 h at room temperature. Then, the membrane was detected using enhanced chemiluminescence reagent (Guan et al., 2017 (link), 2021 (link)).

The cells were treated with the indicated compounds and lysed. Extracted cellular proteins (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was first blocked with Blocking One-P reagent (Nacalai Tesque, Kyoto, Japan) for 30 min at room temperature, and then incubated with the following primary antibodies: rabbit anti-phospho-ERK1/2 (1:1000; Epitomics Inc., Burlingame, CA, USA), rabbit anti-phospho-Akt, rabbit anti-Akt (1:5000; Abcam, Cambridge, UK), rabbit anti-ERK1/2 (1:1000; Cell Signaling Technology, Beverly, MA, USA), or rabbit anti-β-actin (1:1000; Cell Signaling Technology) at 4 °C overnight. This was followed by incubation with peroxidase-linked secondary antibody (1:20000; GE Healthcare, Little Chalfont, UK) at room temperature for 30 min. The labeled proteins were detected with enhanced chemiluminescence using the Prime Western blotting detection system (GE Healthcare).

Western blot analysis was performed as described previously [5 (link)]. Briefly, proteins were separated using SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked in Tris-buffered saline with 5% milk and 0.05% Tween and incubated overnight at 4°C with primary antibodies including rabbit anti-Rab35 (1 : 1000, USA), rabbit anti-PI3K (Abcam), rabbit anti-Akt (Abcam), rabbit anti-mTOR (Abcam), rabbit anti-GFP (Abcam), and rabbit anti-PTPRN2 antibodies (Sigma). After being washed 3-5 times with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Jackson ImmunoResearch) and visualized using enhanced chemiluminescence reagents.