Anti akt

The A549 cells were lysed with RIPA lysis buffer (50mM Tris pH 7.4, 150mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS) and 1% phenylmethylsulfonyl fluoride and a BCA Kit was used to quantify the protein concentrations. The protein samples were resuspended in 5× loading buffer and denatured for 10 minutes at 100°C. Proteins were separated in 6% or 10% SDS-PAGE and transferred onto NC membranes by electroelution (100 V, 50–120 minutes, moist electrotransfer). The membranes were blocked at room temperature for 1 hour using 5% blocking solution and then incubated at 4°C overnight with a 1:1000 dilution of anti-GAPDH, anti-NF-κB p65, anti-Erk1 + 2 (Abcam, Cambridge, UK), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, Beverly, Massachusetts, USA), anti-AKT, and anti-phospho-AKT (Ser473) (Abclonal, Wuhan, China). The membranes were washed with Tris buffer containing 0.1% Tween-20 (TBST) three times, then incubated with secondary antibody (1:5000; Signalway Antibody LLC, Maryland, USA) for 1 hour at room temperature. After several washes with TBST, electrochemiluminescence was used to detect the immunoreactive proteins.

Human AML THP-1 cells were purchased from the Cell Bank of Chinese Academy of Sciences. Matrine was purchased from Tianyuan Biological Agent Plant. The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. RPMI-1640 medium, fetal bovine serum (FBS), dimethyl sulfoxide dissolving (DMSO), penicillin and streptomycin were purchased from Invitrogen; Thermo Fisher Scientific, Inc. The ELISA reader ELx800 was obtained from Bio-Tek Instruments, Inc. Inverted phase contrast microscope was obtained from Olympus Corporation. LY294002 was purchased from Cell Signaling Technology, Inc. and was dissolved in DMSO according to the manufacturer's instructions. Antibodies, including anti-PI3K, anti-phosphorylated (p)-PI3K, anti-mTOR, anti-p-mTOR, anti-Akt, anti-p-Akt and anti-β-actin were purchased from ABclonal Biotech Co., Ltd. The current study was approved by the Ethics Committee of Bengbu Medical College (Bengbu, China).

Briefly, cells or mouse tissues were homogenized in radioimmunoprecipitation assay buffer (PS0012, Leagene). Protein concentrations were determined using the BCA Protein Assay Kit (P0010S, Beyotime). Samples were loaded on 10% SDS–polyacrylamide gel electrophoresis gels and then transferred to nitrocellulose membranes. Immunoblotting was done in primary antibody dilution buffer (A1810, Solarbio) with the corresponding antibodies. The antibodies were purchased as follows: anti–GLP-1R (PA5-97790, Invitrogen), anti–phospho-Akt (AP0304, ABclonal), anti-Akt (A5523, ABclonal), anti–phospho-AMPK (2535S, Cell Signaling Technology), anti-AMPK (5831S, Cell Signaling Technology), anti–phospho–IRS-1 (3203S, Cell Signaling Technology), anti–IRS-1 (3407S, Cell Signaling Technology), and anti–β-actin (4970S, Cell Signaling Technology).