Anti akt

Western blot analysis was performed using an extraction buffer as described [44 (link)]. MKN-45, MKN-28 and U87 cells were cultured in serum-free medium overnight, then treated with the indicated dose of peptide CM 7 for 24 h at 37 °C after stimulated without or with HGF for 15 min. Next, total cell lysates were separated by 12% SDS-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were probed with following primary antibodies: anti-phospho-Met (Tyr1234/1235, (Cell Signaling Technology, Beverly, MA, USA), anti-Met (Affinity, USA), anti-phospho-AKT (Ser473, (Affinity, USA)), anti-AKT (Affinity, USA), anti-phospho-Erk1/2 (Thr202/Tyr204, (Affinity, USA)), anti-Erk1/2 (Affinity, USA), anti-E-cadherin (WanleiBio, Shengyang, China), and anti-GAPDH (Multi Sciences, Hangzhou, China). Then, horseradish-peroxidase-conjugated anti-rabbit IgG was used as a second antibody. The immunoreactive proteins were exposed using an enhanced chemiluminescence detection reagent (WanleiBio, Shengyang, China). After sacrificing the mice and isolating the tumor tissue, Western blot was performed as previously described.

Proteins were extracted from proximal nerve tissue (5 mm long) or SCs transfected with MEG3‐siRNA or control for Western blot analysis as previously described.¹³ The primary antibodies were as follows: anti‐PTEN (Cell Signaling Technology, Danvers, MA, USA) (1:1000), anti‐PI3K (CST, 1:1000), anti‐p‐PI3K (Affinity Biosciences, OH, USA) (1:1000), anti‐AKT (CST, 1:1000), anti‐p‐AKT (CST, 1:1000), anti‐S6 (CST, 1:1000), anti‐p‐S6 (CST, 1:1000) and β‐actin (CST, 1:1000). Anti‐rabbit IgG (CST, 1:2000) was used as the second antibody, and β‐actin was used as the internal control.

According to the instructions of the exosome protein extraction kit (Invitrogen, Carlsbad, CA, USA), isolated exosomes were mixed with a five times Laemmli Sample Buffer (10%  β mercaptoethanol, Bio-Rad, USA), diluted with PBS to acquire an equal concentration of protein. Protein lysis buffer containing PMSF, protease, and phosphatase inhibitors was used to extract total proteins. The target proteins and internal reference protein were separated under the indication of prestained marker proteins, followed by membrane transfer and incubation with specific primary antibodies at 4°C overnight. The antibodies used include Rabbit anti-CD34, anti-CD63 and anti-GAPDH (Abcam), rabbit anti-PTEN, anti-PI3K, anti-p-PI3K, anti-AKT, and anti-p-AKT (Affinity, Cincinnati, OH, USA). Signals were detected using secondary antibodies labeled with IRDye 800 (Rockland Immunochemicals, Gilbertsville, PA) and signal intensities were determined using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Doxorubicin (Dox) purchased from Sigma‐Aldrich was dissolved in sterile double distilled water at a concentration of 3 g/L before experiment. Anti‐CrkL, anti‐p‐AKT, anti‐AKT, anti‐MRP1, and anti‐β‐actin monoclonal antibodies were purchased from Affinity Biosciences and Abcam Inc. MTT and Annexin Ⅴ‐FITC/PI Kit were obtained from 4A biotech. The human myelogenous leukemia K562 cells doxorubicin‐resistant counterpart K562/ADR cells were obtained from the Tongpai Biotechnology CO., LTD. K562 cells were cultured in RPMI‐1640 medium containing 10% fetal bovine serum and incubated at 37˚C in a 5% CO₂ atmosphere. The culture medium of K562/ADR cells required an additional supplement of 1 mg/L Dox. K562/ADR cells need to be cultured in Dox‐free medium for 2 days before the experiments.

Total proteins were extracted from cultured cells and adipose tissue with protein lysis buffer RIPA (Goodbio Technology, China) supplemented with the protease inhibitor PMSF and a phosphatase inhibitor according to the manufacturer’s protocol. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene difluoride membrane before immunoblotting. The following primary detection antibodies used in this study: anti-SIRT1 (Cell Signaling Technology CST, No. 2028, USA), anti-phospho-AKT (Ser 307) (CST, No. 12694S, USA), anti-AKT (Affinity Biosciences, AF6261, USA), anti-p-ERK1/2 (Thr 202/Tyr 204) (Affinity Biosciences, AF1015), anti-ERK1/2 (Affinity Biosciences, AF0155), anti-phospho-JNK1/2/3 (Thr183+Tyr185) (Affinity Biosciences, AF3318, USA), anti-JNK (Santa Cruz Biotechnology, sc-7345, USA) and anti-β-actin (Santa Cruz Biotechnology, sc-47778, USA). Anti-mouse or anti-rabbit IgG-HRP (Invitrogen, USA) were used as secondary detection antibodies. Finally, immunoreactive bands were detected using the Super Signal chemiluminescence detection kit (Thermo Scientific, USA) in imaging system FluorChem M apparatus (CareStream 2200 PRO, USA). β-actin used as an endogenous control. The density of the bands was analyzed using image analysis software (IMAGE J).

Acacetin (purity 98%) was purchased from Tauto Biotech Co., Ltd. (Shanghai, China). RPMI 1640 medium was obtained from Gibco (Beijing, China). Foetal bovine serum was purchased from Gibco (CA, USA). Cell Counting Kit-8 (CCK-8) reagent was purchased from Dojindo (Dojindo Laboratories, Japan). TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). Anti-p-PI3K (Tyr458), anti-p-Akt (Ser473), anti-PI3K and anti-Akt were purchased from Affinity Biosciences. Anti-MMP-9, anti-MMP-2 and anti-Snail were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Vimentin, anti-N-cadherin, anti-E-cadherin, anti-GAPDH, and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Proteintech (Wuhan, China).