Anti p pi3k

After washing with PBS, the cells were lysed with prechilled lysis buffer. The collected cell lysate was centrifuged at 14000 × g for 15 min at 4°C, and the protein content was measured with 5 × sample buffer (BSA; Thermo Fisher Scientific, Inc., CA, USA). Then, these protein samples were tested by WB. The 4%-20% precast gel was used to transfer the protein to the nitrocellulose membrane, and the 5% skimmed milk was used to seal the membrane at ambient temperature (Bio-Rad Laboratories) 1 h. Subsequently, specific antibodies (diluted 1 : 10 00), including anti-FGF12 (ab231956, Abcam), anti-E-cadherin (ab231303, Abcam), anti-Vimentin (ab8978, Abcam), anti-p-PI3K(#17366, Cell Signaling Technology), anti-PI3K(#4249, Cell Signaling Technology), anti-p-AKT(S473)(#4060, Cell Signaling Technology), anti-N-cadherin(ab18203, Abcam), anti-p-AKT(T308)(#13038, Cell Signaling Technology), anti-AKT(#4685, Cell Signaling Technology), and anti-β-actin (#4970, Cell Signaling Technology), were used to block membranes. Subsequently, the secondary antibody was used to incubate the membranes (Santa Cruz) at ambient temperature for 1 h and visualized them using ECL solution (Bio-Rad Laboratories). Finally, a chemiluminescence imaging system (Mini HD9; UVitec, Cambridge, UK) was used to take images.

Protein was extracted from MGC-803 cells at different groups and tumor tissues using RIPA buffer containing PMSF. Proteins (50 µg) were resolved on 10% SDS-PAGE and electrotransferred on polyvinylidene fluoride membranes (Invitrogen, CA, USA). The primary antibodies used here were anti-PI3K, anti-p-PI3K, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-p53, anti-CDK4, anti-CyclinD1 or anti-beta-actin from Cell Signaling Technology or Abcam. Then, the membranes were incubated with secondary antibody and target proteins were analyzed by ImageLab software (Bio-Rad, CA, USA) [25 (link)].

The following antibodies were used: mouse monoclonal anti-PARP-1, mouse monoclonal anti-BrdU (#347580), mouse monoclonal anti-p21 (#556431), mouse monoclonal anti-p27 (#610241), rabbit polyclonal anti-PAR (#551813,; BD Bioscience), mouse monoclonal anti-α-tubulin (#T5168, Sigma-Aldrich), mouse monoclonal antibodies for Myc (#G019), HA (#G036), Flag (#G191), and GFP (#G096, Abm), rabbit polyclonal anti-MAP2 (#MAB3418), anti-Olig2 (#ab9610), anti-SOX2 (#ab5603, Millipore), anti-GFAP (#ab7260), anti-Ki67 (#ab15580), anti-Tbr2 (#ab183991) and mouse monoclonal anti-Nestin (#ab22035, abcam), goat polyclonal anti-DCX (#sc-8066), anti-NeuroD (#sc-1084), rabbit polyclonal anti-ERK (#sc-93), mouse monoclonal anti-pERK (#sc-7383), anti-LaminB1 (#sc-56145, Santa Cruz), rabbit polyclonal anti-Akt (#4060), rabbit polyclonal anti-pAkt (#4061), rabbit polyclonal anti-PI3K (#4228), anti-pPI3K (#4257), rabbit polyclonal anti-FOXO1 (#2880), rabbit polyclonal anti-pFOXO1 antibody (#9461, Cell Signaling).

The Qingrekasen Granule was purchased from Xinjiang Uygur Pharmaceutical Co., Ltd. (NO.1807521, Xinjiang, China). Adriamycin (ADR) was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (No. 2007E1, Shenzhen, China), and Benazepril was purchased from Novartis (No. 2007, Beijing, China). The BCA Protein Assay Kit was obtained from Beyotime Biotech Inc. (P0012, Shanghai, China), and Guangzhou Jet Bio-Filtration Co., Ltd. (Guangzhou, China) provided the Elisa plates (12 well strip ×8). HPLC grade methanol and acetonitrile were purchased from Merck KGaA (Darmstadt, Germany). Anti-PI3K and anti-p-PI3K antibodies were purchased from Cell Signaling Technology Inc. (MA, United States). The Anti-AKT, anti-p-AKT, anti-mTOR, anti-P-mTOR, anti-BCL-2, anti-Caspase-3, and Beta-actin antibodies were purchased from Bioworld Technology Inc. (MN, United States). All other reagents were HPLC grade.

SGC-7901 and BGC-823 cells were seeded in a 6-well culture dish, after which they were treated with RPMI 1640 medium containing 28-hydroxy-3-oxoolean-12-en-29-oic acid (40, 80 and 160 μmol/L) for 24 h and cultured for 24 h. Subsequently, cells were lysed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer using standard methods. Protein lysates were separated on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk for 2 h at room temperature. Membranes were incubated with primary antibody followed by HRP-conjugated anti-IgG at room temperature for 2 h. Signals were visualized by enhanced chemiluminescence (ECL). A gel imaging analysis system (Bio-Rad) was used to detect the protein bands. The antibodies, including rabbit anti-β-actin, anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-MMP-2, anti-MMP-9, anti-AKt, anti-phosphorylated (p)-Akt, anti-PI3K, anti-(p)-PI3K, and anti-Snail were purchased from Cell Signaling Technology (Beverly, MA, USA). HRP-labeled goat anti-rabbit IgG was purchased from Hangzhou Huaan Biotechnology Co.

Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), and other materials for cell culture were purchased from Gibco BRL (Carlbad, CA, USA). D-[3-³H] deoxy-glucose (20 Ci/mmol) was purchased from Perkin-Elmer (Boston, MA, USA). Cytochalasin B (Cyt B), 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES), S100B protein, anti-S100B antibody (SH-B1), o-phenylenediamine (OPD), Anti-S100B antibody (clone SH-B) and antibodies for blotting: anti-p PI3K was purchased from Cell Signaling (Frankfurt, Germany); anti-p p38 and anti-GLUT2 were from Sigma-Aldrich (St. Louis, MO, USA); anti-insulin and anti-p STAT3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and anti-β-actin and anti-p IRS were from EMD Millipore (Darmstadt, Germany). Other reagents were purchased from local commercial suppliers (Sulquímica, Labsul or Biogen; Porto Alegre, Brazil).