Phosphor pi3k

Tetrandrine (Tet) (C₃₈H₄₂N₂O₆) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Tet was solubilized in 0.1 M HCl to a concentration of 25 mg/mL as the stock solution and then diluted to the desired concentrations before use. Antibodies against PI3K, phosphor-PI3K, PDK1, p-PDK1, Akt, phospho-Akt, NF-κB, and MMP-9 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). LY294002 (PI3K inhibitor), PDTC (NF-κB inhibitor), TNF-α (NF-κB activator) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). SC79 were purchased from abcam. Inc.(Cambridge, Britain).

Whole cell lysates of NPC cells were prepared with a proteinase and phosphatase inhibitor cocktail (Roche, CA, USA). Equal amounts of proteins were resolved to 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Danvers, MA, USA) and blocked with 5% nonfat dry milk in Tris-buffered saline, pH 7.5. Membranes were immuno-blotted overnight at 4 °C. Protein blots were probed with primary antibodies against HIPK2 (Abcam #ab108543), E-cadherin (Cell Signaling Technology #3195), N-cadherin (Cell Signaling Technology #13116), SPEN (Abcam #ab72266), c-JUN (Abcam #ab40766), phosphor-AKT (Cell Signaling Technology #4060), AKT (Cell Signaling Technology #4691), phosphor-PI3K (Cell Signaling Technology #17366), and PI3K (Cell Signaling Technology #4292). Goat anti-rabbit IgG HRP-linked antibody (1:10,000) and goat anti-mouse IgG HRP-linkedantibody (1:10,000) were from Proteintech (Rosemont, IL, USA). Signals were visualized by chemiluminescence (Bio-rad, Hercules, California) and quantitated using a Quantity One system (Bio-Rad, Hercules, CA, USA).

The activation of the PI3K/AKT and NF-κB signaling pathways in colon tissue was measured by western blotting. The total protein of colon tissues was extracted using RIPA lysis buffer containing a protease inhibitor cocktail (Beyotime Biotechnology, China), and protein concentrations were measured using a Bradford protein assay kit (Bio-Rad, USA). The proteins were separated by SDS-PAGE and then electrotransferred to PVDF membranes. The membranes were then blocked with 5% nonfat skim milk and incubated with primary antibodies overnight at 4°C. Then, membranes were washed with TBST and incubated with secondary antibody (goat anti-rabbit IR Dye 800, Invitrogen) for 1 hour at room temperature, followed by visualization with an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE) and analysis using Quantity One image analysis software. GAPDH was used as an internal control to normalize the expression of protein. The antibodies used were as follows: PI3K (1 : 1000; Cell Signaling Technology), phosphor-PI3K (1 : 1000; Cell Signaling Technology), AKT (1 : 1000; Abcam), p-AKT (1 : 800; Abcam), NF-κBp65 (1 : 800; Abcam), p-IκBα (1 : 800; Abcam), IκBα (1 : 800; Abcam), IKKα (1 : 800; Abcam), p-IKKα (1 : 800; Abcam), and GAPDH (1 : 1000; Cell Signaling Technology).

The NP460 cell line (human normal nasopharyngeal epithelial cell) was kindly provided by Professor Tsao’s research group from Hong Kong University. The cells were cultured in 1:1 ratio of Defined Keratinocyte-SFM (DKSFM) medium and Epilife medium supplemented with growth factors EDGS (Gibico). The human nasopharyngeal carcinoma cell line 5-8F was cultured as previously described (23 (link)). The potentially high metastasis nasopharyngeal carcinoma cell line S18 was kindly provided by Professor Qian of the Cancer Center of Sun Yat-sen University. Cells were cultured in DMEM (Hyclone) supplemented with 10% Fetal Bovine Serum (FBS) (Gibico) and 1% Penicillin-Streptomycin solution (Hyclone). All cell lines were cultured at 37°C in a humidified incubator with 5% CO₂.
Reagents were sourced commercially as follows: YBX3 antibody was purchased from Immuno-Biological Laboratory; GAPDH, AKT, phospho-AKT, E-cadherin, Vimentin, MMP1 antibodies were purchased from Protein Technology; PI3K, phosphor-PI3K were purchased from Cell Signaling Technology.

Western blot analysis was performed according to conventional methods. Antibodies against Akt, phosphor-Akt, PI3K, phosphor-PI3K, and GAPDH were from Cell Signaling Technology.