Western blotting analysis was performed according to a previous description46 (link). Antibodies included anti-PTEN, CDK1, p21, β-catenin, N-cadherin, E-cadherin, TCF4, c-Myc, c-JUN, AKT, p-AKT (Ser473), PI3K, p-PI3K (Tyr458), Ras, p-ERK, and p27 antibodies (1:1000; Cell Signaling Technology, Danvers, MA, USA) and ZEB2, CDK4, and β-actin antibody (1:500; Santa Cruz Biotechnology). Images were captured with ChemiDocTM CRS+Molecular Imager (Bio-Rad).
P pi3k tyr458
Manufactured by Cell Signaling Technology
Sourced in United States
P-PI3K (Tyr458) is a laboratory reagent that detects the phosphorylation of the p85 regulatory subunit of PI3 kinase at tyrosine 458. It is used as a research tool to study the activation of the PI3K/AKT signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
P akt ser473, by Cell Signaling Technology (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Chemidoc crs molecular imager, by Bio-Rad (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
C jun, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Mgc 803, by American Type Culture Collection (1 mentions)
Sgc 7901, by American Type Culture Collection (1 mentions)
Ges 1, by American Type Culture Collection (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Bgc 823, by American Type Culture Collection (1 mentions)
Hgc 27, by American Type Culture Collection (1 mentions)
β actin, by Proteintech (1 mentions)
Rgm 1, by American Type Culture Collection (1 mentions)
Wnt974, by MedChemExpress (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
4 protocols using p pi3k tyr458
1
Western Blot Analysis of Key Signaling Proteins
2
Gastric Cancer Cell Lines and Antibodies
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The human gastric cancer cell lines, including SGC‐7901, MGC‐803, HGC‐27 and BGC‐823, and two human gastric epithelial cell lines, such as GES‐1 and RGM‐1, were obtained from the American Type Culture Collection (ATCC). All cells were maintained in RPMI 1640 (GIBCO BRL, Grand Island, NY), supplemented with 10% FBS (GIBCO BRL, Grand Island, NY) and cultured at 37°C in 5% CO2 incubator. The antibodies were purchased from Sigma (β‐actin), Proteintech (GHR) and Cell Signaling Technology (PCNA, p‐PI3K‐Tyr458, P‐AKT‐Ser473, PI3K, AKT, CDK4, Cyclin D1, Cleaved‐PARP, IR DyeR 680 goat anti‐Mouse, IR DyeR 800 goat anti‐Mouse).
3
Artesunate and WNT974 Signaling Pathway
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Artesunate (ART) was provided from Kuming Pharmaceutical Co. Ltd. WNT974 was purchased from MedChemExpress company, Dulbecco's Modified Eagle's Medium, fetal bovine serum were purchased from Thermofisher Scientific Company. Antibodies against β-TrCP, GSK-3β, ANAPC2, GSK-3β, p-Akt (Ser473), t-Akt, p-PI3K (Tyr458), t-PI3K, p-mTOR (Ser2448), t-mTOR and β-Actin were purchased form Cell Signaling Technology (Danvers, MA). Antibodies against KRAS, NRAS, HRAS were purchased form abcam (USA). Mouse anti-rabbit IgG-HRP secondary antibody was purchased from San Cruz Biotechnology (Santa Cruz, United States of America).
4
Western Blot Analysis of EMT Markers
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RIPA lysis buffer containing 1% PMSF (Beyotime, China) was used to collect total protein from cell lines. 10% SDS-PAGE was used to separate protein and PVDF membranes were loaded with separate protein. All membranes were blocked with 5% degreasing milk and incubated with primary antibodies: MSMO1 (Abcam), E-cadherin (Abcam), β-catenin (Proteintech), Vimentin (Proteintech), AKT (Bimake), PI3K (Bimake), m-TOR (Bimake), p-AKT (Ser473) (Cell Signaling Technology), p-PI3K (Tyr458) (Cell Signaling Technology), p-mTOR (Ser2448) (Cell Signaling Technology), GAPDH (Proteintech) overnight at 4 °C. Next day, membranes were incubated with secondary antibodies for two hours. Finally, blots were detected by ECL kit (Beyotime, China). The experiments were done thrice.
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