Anti phospho akt p akt

Anti-PBK/TPOK mouse polyclonal antibody (sc-293028) and anti-ACTB antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PDZ-binding kinase/T-LAK cell-originated protein kinase is an affinity purified mouse polyclonal antibody raised against a recombinant protein of PBK/TOPK. Anti-p53 (DO7) antibodies were from Novocastra Laboratories (Newcastle upon Tyne, UK), and anti-p53 (DO1) and anti-p21 were from Santa Cruz Biotechnology. Anti-PTEN, anti-AKT and anti-phospho-AKT (pAKT) were from Cell Signaling Technology (Cell Signaling, Massachusetts, USA). Cells were lysed and their proteins were extracted by M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, Waltham, MA, USA).

HUVECs were seeded and incubated at a density of 2 × 10⁵ cells in 60 mm dishes for 2 days. Culture medium was then replaced with fresh, reduced serum medium (2% FBS) and different concentrations of murrangatin for 24 h. Cellular proteins were harvested and immunoblots were performed as previously reported.9 (link) The antibodies used included anti-AKT (catalog# 9272; Cell Signaling Technology, Danvers, MA, USA), antiphospho-AKT (P-AKT, catalog# 9271; Cell Signaling Technology), anti-total ERK1/2 (T-ERK1/2, catalog# 4695S; Cell Signaling Technology), anti-phospho-ERK1/2 (P-ERK1/2, catalog# 9102; Cell Signaling Technology), and anti-β-actin (catalog# 2565; Biosynthesis Biotechnology, Beijing, China).

Western blot analysis was performed as previously described [22] (link). After sample preparation, equal amounts of a sample protein (50 μg) were loaded onto an SDS-PAGE gel. After electrophoresis, the samples were transferred onto a nitrocellulose membrane. Then, the membrane was blocked for 2 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-apoE (1:500, Abcam, Cambridge, MA, USA), anti-Iba-1 (1:1000, Wako, USA), anti-APP (1:1500, Abcam, Cambridge, MA, USA), anti-myelin basic protein (MBP, 1:1000, Abcam, Cambridge, MA, USA), anti-LRP1 (1:1000, Abcam, Cambridge, MA, USA), anti-Shc1 (1:2000, Abcam, Cambridge, MA, USA), anti-PI3K (1:1000, Cell signaling, USA), anti-Akt (1:1000, Cell signaling, Danvers, MA, USA), anti-phospho-Akt (p-Akt, 1:1000, Cell signaling, Danvers, MA, USA), anti-CD16 (1:1500, Santa Cruz, Dallas, TX, USA), anti-iNOS (1:500, Abcam, Cambridge, MA, USA), anti-CD206 (1:1500, Santa Cruz, Dallas, TX, USA), and anti-β-actin (1:5000, Santa Cruz, Dallas, TX, USA). Appropriate secondary antibodies (1:5000, Santa Cruz, Dallas, TX, USA) were selected to incubate with the membrane for 2 h at room temperature. Then, blot bands were visualized with an ECL reagent (Amersham Biosciences UK Ltd., PA, USA). Non-saturated bands were selected to perform densitometry quantification using Image J software (Image J 1.51, NIH, USA).