Pi3 kinase p85

Eluted immune complexes were resolved in precast gradient NuPAGE 4–12% Bis-Tris gels (Invitrogen) and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked with 2% BSA and incubated overnight at 4 °C with primary antibodies, including; Gab2 (sc-9313), SHP2 (sc-7844) and 14-3-3 (sc-25276) from SantaCruz Biotechnology, ARID3A (A300-228 A) and ARID3B (A302-564 A) from Methyl Lab, GRB2 (610111, Transduction Lab), PI3 kinase p85 (#4257, Cell Signaling) and DLAT (ab126224, Abcam). Then, the membranes were repeatedly washed with TBST buffer (150 mM NaCl, 10 mM Tris, pH 8.0 and 0.1% Tween 20) and incubated with HRP-conjugated anti-rabbit (NA934, GE Healthcare), anti-mouse (NA931, GE Healthcare) or anti-goat (ref) secondary antibodies for 1 h at room temperature. Finally, the membranes were washed and processed with ECL solution (PRN2106, GE Healthcare) for detection scanning on a Kodak Image Station 1000. The intensity of the bands was quantified using ImageJ software (National Institutes of health, Bethesda, MD, USA).

The MTT cell proliferation assay, propidium iodide, ribonuclease (RNase A), protease inhibitor cocktail, radioimmunoprecipitation assay buffer, bovine serum albumin, and Dulbecco’s phosphate buffered saline (PBS) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Human ZIP4 antibodies were purchased from Proteintech (Rosemont, IL). PCNA and phospho-PI3 kinase p85 (Tyr607) antibodies were purchased from Abcam (Cambridge, MA, USA). Cleaved caspase-3, cleaved caspase-9, Bax, Bcl-2, E-cadherin, FSP-1, Vim, PI3 kinase p85, Akt, phospho-Akt (Ser473), phospho-Akt (Thr308), GSK3β, and phospho-GSK-3β (Ser9) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Human β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). All antibodies were used according to the manufacturers’ instructions. PI3K inhibitors (LY294002 and wortmannin) were purchased from Sigma-Aldrich Co. The other chemicals used in this study were of analytical reagent grade.

A radio-resistant cell line (MDA-MB-231/IR) was recently developed in our laboratory [12 (link)]. Cell culture media [Dulbecco’s modified Eagle’s medium (DMEM)], fetal bovine serum (FBS), and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA). 10-gingerol (Item number: 11842; ≥98% purity) was purchased from Cayman chemicals (Ann Arbor, MI, USA). All other chemicals used in experiments were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. Primary antibodies caspase-3, cleaved-caspase-3, caspase-7, Bax, Bcl-2, PARP, cyclin D1, GAPDH, PI3 Kinase p85, p-PI3 Kinase p85α, Akt, p-Akt (Ser473), p-Akt (Thr308), mTOR, p-mTOR, 4E-BP1, p-4E-BP1, caveolin-1, GSK-3β, and p-GSK-3β were purchased from Cell Signaling Technology (Beverly, MA, USA). β-catenin was purchased from BD bio-sciences (San Diego, CA, USA). HRP-conjugated secondary antibodies (goat anti-rabbit and anti-mouse IgG) were purchased from Vector Laboratories (Burlingame, CA, USA). An ECL Plus kit (western blotting substrate) was purchased from Biosesang Inc. (Seongnam, Korea).

Total protein was extracted from liver tissue, using RIPA protein lysis buffer containing 1 mM PMSF. 300 ng total protein were separated by 10% SDS-PAGE gel at 80 V for 1 h, transfered with polyvinylidenedifluoride (PVDF) membrane at 100 V for 1.5 h, blocked in 5% BSA (Solarbio, Beijing, China) and probed with appropriate primary antibodies against the target proteins. PTEN (Cat:#9188S), PI3 Kinase p85 (Cat:#4292S) anti bodies were purchased from Cell Signaling (Beverly, MA, USA). p-Akt (Cat:66444-1-Ig) and β-actin (Cat:20536-1-AP) anti bodies were purchased from Proteintech (Protein tech, Chicago, IL, USA). These were followed by incubation with Goat Anti-Rabbit immunoglobulin (Ig) G (H+L) (Cat:SA00001-2) (Protein tech, Chicago, IL, USA), and antigen-antibody complexes were visualized using the chemilucent ECL (TransGen Biotech, Beijing, China) detection system. The densitometric analysis conducted by Image J software 1.6.0 (National Institutes of Health, Bethesda, MD, USA) [37 (link),38 (link),39 (link)].

The human hepatoma cell line SMMC-7721 was plated in 6-well plates at a density of 5 × 10⁴ per well. After overnight culture, the TF at concentration of 1.0 mg/mL was added to the wells and cells were incubated for 48 h. Protein extracts were isolated from each group cells in using RIPA protein lysis buffer containing 1 mM PMSF. Total protein was separated by 10% SDS-PAGE, transferred with polyvinylidenedifluoride (PVDF) membrane, blocked in 5% BSA (Solarbio, Beijing, China), and probed with appropriate primary antibodies against the target proteins. PTEN, PI3 Kinase p85, p-Akt, andβ-actin antibodies were purchased from Cell Signaling (Beverly, MA, USA). These were followed by incubation with Goat Anti-Rabbit immunoglobulin (Ig) G (H + L) (Protein tech, USA), and antigen-antibody complexes were visualized using the chemilucent ECL (TransGen Biotech, China) detection system. The densitometric analysis was conducted by using custom Image J-based software, as reported earlier [19 (link), 20 (link)].

Western blot analyses were performed with precast gradient gels (Bio-Rad) using standard methods. Briefly, cells were scraped from flasks and treated with PBS, DV1-N₃ solution (30 μM), L-DBCO, L-s-DV1-24k, L-DV1-9k, L-DV1-24k, L-DV1-39k, L-DV1-54k, and L-DV1-73k for 1 h on the ice bath. Washed the cell suspension with PBS by centrifuge. After that lysed cell samples in the radio immunoprecipitation assay (RIPA) buffer containing protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were probed with the specific primary antibodies: antibodies to β-actin (1:5000, 4967S), p115 RhoGEF (D25D2) (1:1000, 3669S), p-PI3K p85(Y458)/p55(Y199) (1:1000, 4228P), PI3 Kinase p85 (1:1000, 4292S) (Cell Signaling Technology), and then with peroxidase-conjugated secondary antibodies Amersham ECL Mouse IgG (GE Health life Science). The bands were visualized by chemiluminescence with Immobilon™ western chemiluminescent HRP substrate (EMD Millipore). ImageJ was used for densitometric analyses of western blots, and the quantification results were normalized to the loading control.