Anti p akt

Cells were lysed with RIPA lysate (Beyotime, Shanghai, China), and the concentration of protein was determined by the BCA assay kit (Solarbio, Beijing, China). Briefly, the equal amount of protein was separated with 10% SDS-PAGE (Epizyme, Shanghai, China), and transferred to PVDF membranes (Millipore, MA, USA). The PVDF membranes were blocked with 5% skim milk and incubated at 4 °C overnight with primary antibody, including anti-Fibronectin (Cell Signaling Technology, MA, USA), anti-N-cadherin (CST), anti-E-cadherin (CST), anti-MMP2 (CST), anti-MMP9 (CST), anti-Cytokeratin (Proteintech, Wuhan, China), anti-Vimentin (CST), anti-Ran (Proteintech), anti-Akt (CST), anti-p-Akt (CST), GSK3β (CST), anti-p-GSK3β (CST), anti-β-catenin (CST). β-actin (CST) was used as internal controls. Then the PVDF membranes were incubated with a secondary antibody for 1 h. The blots were scanned by Amersham Imager 600 (GE, Boston, MA, USA). Each sample was repeated three times and the three independent western blot bands were quantitatively analyzed by Image J software (NIH, Bethesda, Maryland, USA).

Extracts from three species of rhubarb (132.8, 265.5, 531 μg/mL) were added in EA.hy926 cells treated by LPS (1 μg/mL). After 48-h incubation, the cells were lysed in RIPA buffer containing the mixture of protease inhibitor and PMSF(#C1055, Beijing, China), and homogenates were centrifuged at 12000 rpm for 5 min at 4°C, and then the supernatant was harvested. According to the protocol, the protein denaturation was carried out after the protein concentration was determined. Equal amounts of protein were subjected to 8% SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with anti-p65, anti-AKT, and anti-STAT3 (1:1500, Proteintech, USA); anti-p-p65 and anti-p-PI3K(1:1000, CST, USA); anti-PI3K, anti-eNOS, and anti-iNOS (1:1000, Proteintech, USA); anti-p-AKT(1:2000, Proteintech, USA); and anti-p-STAT3 (1:2000, Abcam, USA), at 4°C overnight, and then the membrane was incubated with secondary antibody and finally added chemiluminescence solution for development.

The total content of cellular protein harvested from RCC cells were extracted using RIPA lysis buffer (Keygen, Nanjing, China) and the protein amounts was determined using a BCA kit (Keygen, Nanjing, China). SDS-PAGE separated same volumes proteins of each lane, then blotted onto PVDF membranes. Primary antibodies were incubated overnight at 4 °C, and HRP-labeled secondary antibody (ABclonal, 1:5,000) 2 hours at RT after blocked with 5% BSA. Signal of immunoreactivities were photographed by ECL reagent (NCM Bio, China) on Tanon 5200 automatic chemiluminescence imaging system (Tanon, China). Antibody listed as follow: Anti-E-cadherin (BD Biosciences, 610181), Anti-Vimentin (Proteintech, 10366-1-AP), Anti-N-cadherin (BD Biosciences, 610920), Anti-PI3k (Proteintech, 67121-1-Ig), Anti-AKT (Proteintech, 101762-2-AP), Anti-p-AKT (Proteintech, 66444-1-Ig), Anti-p-mTOR (Cell Signaling Technology, 5536S), Anti-Bcl-2 (Cell Signaling Technology, 155071S), Anti-Cleved-Caspaes 9 (Cell Signaling Technology, 20750S), Anti-p53 (Proteintech, 10442-1-AP), and we took GAPDH(Santa Cruz, sc-32233) as control.

The total protein was extracted from A549 cells using RIPA buffer containing protease inhibitors and separated through Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Then, proteins were transferred to a PVDF membrane. After blocked with 5% fat‐free milk for 1 hour, the sample was incubated with primary antibodies for 1 hour and then with secondary antibodies for 1 hour. The following primary antibodies, namely anti‐Syncytin 1 (1:500), anti‐SP1 (1:1000) anti‐Active‐Caspase3 (1:1000), and anti‐Active‐Caspase9 (1:1000) were purchased from Abcam; anti‐Cyclin D1 (1:1000), anti‐Nusap1 (1:1000), anti‐CDK6 (1:1000), anti‐CDK4 (1:1000), anti‐Bcl2 (1:1000), anti‐Bax (1:1000), anti‐β‐catenin (1:1000), anti‐Vimentin (1:1000), anti‐E‐cadherin (1:1000), anti‐N‐cadherin (1:1000), anti‐P70 (1:1000), and anti‐GAPDH (1:5000) were purchased from Proteintech Group; anti‐p‐AKT (1:1000), anti‐p‐mTOR (1:1000), and anti‐p‐Erk1/2 (1:1000) were purchased from Cell Signaling Technology.

Total protein was extracted with RIPA lysis buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to polyvinylidene difluoride membranes, blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, and then incubated with anti-PI3K, anti-AKT, anti-p-PI3K, anti-p-AKT, and anti-β-actin primary antibodies (Proteintech, Rosemont, IL, USA) for 16 h at 4°C. Membranes were washed three times in Tris-buffered saline containing 0.1% Tween-20 and then incubated with anti-mouse or anti-rabbit IgG secondary antibodies for 1 h. Immunoreactive bands were visualized by a commercial electrochemiluminescence (ECL) kit (Amersham Pharmacia Biotech, Little Chalfont, Great Britain).

Cells and tissues were harvested and lysed in RIPA buffer for 30 min at 4-8ºC. Proteins were detected and quantified using BCA protein assay kit (Thermo Fisher Scientific). Protein samples (30-50 μg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Nonspecific binding sites were blocked with 5% low-fat milk and 0.1% Tween-20 at room temperature for 2 h. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Proteintech (Wuhan, China): anti-SSRP1 (1:1000; 15696-1-AP), anti-p27 (1:1000; 25614-1-AP), anti-p21 (1:1000; 10355-1-AP), anti-CyclinD1 (1:1000; 60186-1-Ig), anti-BCL2 (1:1000; 26593-1-AP), anti-BAX (1:1000; 50599-2-Ig), anti-MDM2 (1:1000; 19058-1-AP), anti-p53 (1:1000; 10442-1-AP), anti-AKT (1:1000; 60203-2-Ig), anti-β-actin (1:2000; 66009-1-Ig) and arigo (Taiwan, China): anti-P-AKT (1:1000; ARG51559); secondary antibody: goat anti-Mouse (1:2000; 10828-I-AP) and goat anti-Rabbit IgG (H+L) (1:2000; SA0000I-2) from Proteintech Group Inc. The membranes were scanned for statistical analysis by enhanced chemiluminescence (ECL) using a gel image processing system (Tanon, Shanghai) and the density of the bands was analyzed using Image J ( Wayne Rasband National Institutes of Health, USA).