Anti phospho pi3k

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-phospho-PI3K is a primary antibody that specifically recognizes the phosphorylated form of the PI3K (Phosphoinositide 3-kinase) protein. PI3K is a critical signaling enzyme involved in various cellular processes, including cell growth, proliferation, and survival.

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Lab products found in correlation

Close spelling variants of this product

Anti-PI3K (35 citations) Phospho-PI3K (4 citations) Anti-phospho-PI3K(Tyr485) (sc-130211) (2 citations)

10 protocols using anti phospho pi3k

Protein Expression and Phosphorylation Profiling

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Cells were harvested and lysed with RIPA buffer (Thermo Scientific Inc., Boston, MA, USA) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and a phosphatase inhibitor cocktail (Sigma-Aldrich). Total protein concentrations were quantified using an RC/DC protein assay reagent (Bio-Rad, Hercules, CA, USA). Protein extracts were separated using 6 and 10% SDS-PAGE and transferred onto PVDF membranes (PALL, Westborough, MA, USA). Membranes were incubated in a blocking solution (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA) for 30 min and incubated with anti-VEGF-A, anti-VEGFR-1, anti-VEGFR-2, anti-phospho FAK, anti-phospho ERK1/2, anti-phospho PI3K, anti-phospho AKT, anti-phospho JNK, anti-phospho p38 antibodies (Santa Cruz Biotech. Inc.) at 1:2000 dilution in a blocking solution overnight at 4 °C, and probed with peroxidase conjugated secondary antibodies at 1:5000 dilution. Protein bands were detected using enhanced chemiluminescent Western blotting detection reagent (Thermo Scientific Inc.)
Also, protein extracts were immunoprecipitated using a mouse anti-phospho-Tyr antibody (Santa Cruz Biotech. Inc.) and an ImmunoCruz™ IP/WB Optima kit (Santa Cruz Biotech. Inc.). Immunoprecipitated proteins were subjected to 6% SDS-PAGE and Western blotting using anti-VEGFR-1 and anti-VEGFR-2 antibodies (Santa Cruz Biotech. Inc.)

Bae M.G., Hwang-Bo J., Lee D.Y., Lee Y.H, & Chung I.S. (2021). Effects of 6,8-Diprenylgenistein on VEGF-A-Induced Lymphangiogenesis and Lymph Node Metastasis in an Oral Cancer Sentinel Lymph Node Animal Model. International Journal of Molecular Sciences, 22(2), 770.

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Antibody-based Signaling Pathway Analysis

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Anti-phospho-PI3K, anti-PI3K, anti-laminB antibody, anti-phospho-PDGF-α^T720, anti-PDGF-α, anti-phospho-PDGF-β^T1009, and anti-PDGF-β antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-HIF-1α antibody was purchased from BD Biosciences (San Jose, CA, USA). Anti-PTEN, anti-phospho-mTor^S2448, anti-mTOR, anti-phospho-Akt^S473, anti-phospho-Akt^T308, anti-Akt, anti-phospho-ERK1/2^T202/T204, and anti-ERK1/2 antibodies were purchased from Cell Signaling (Beverly, MA, USA). Anti-brachyury antibody was purchased from R&D Systems. Anti-mouse-IgG-HRP and anti-rabbit-IgG-HRP secondary antibodies were purchased from Santa Cruz Biotechnology. The PDGFR inhibitor, 3-Fluoro-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenylamine, was obtained from EMD Chemicals (Gibbstown, NJ, USA, catalog #521233). A concentration of 1 μM was used for these studies. LBH589 was obtained from Selleck (Houston, TX, USA, catalog #S1030).

Lee D.H., Zhang Y., Kassam A.B., Park M.J., Gardner P., Prevedello D., Henry S., Horbinski C., Beumer J.H., Tawbi H., Williams B.J., Shaffrey M.E., Egorin M.J., Abounader R, & Park D.M. (2015). Combined PDGFR and HDAC Inhibition Overcomes PTEN Disruption in Chordoma. PLoS ONE, 10(8), e0134426.

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Western Blot Analysis of Protein Targets

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Total protein was extracted from frozen tissues or cells using RIPA lysis buffer (Beyotime, Nantong, P.R. China). A total of 30 μg of protein samples from each cell line was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat dry milk in 0.05% Tween 20 phosphate-buffered saline for 1 h at room temperature, membranes were incubated with the corresponding primary antibodies (mouse anti-JARID2, anti-E-cadherin, anti-N-cadherin, anti-phospho-PI3K, anti-PI3K, anti-phospho-Akt, anti-Akt, or anti-GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Subsequently, membranes were rinsed and incubated with goat anti-mouse horseradish peroxidase-conjugated IgG (Santa Cruz Biotechnology). Bound antibodies were detected using enhanced chemiluminescence (ECL) reagents (Pierce, Rockford, IL, USA).

Li Z., Xu C., Gao M., Ding B., Wei X, & Ji N. (2017). Reduced Expression of Jumonji AT-Rich Interactive Domain 2 (JARID2) in Glioma Inhibits Tumor Growth In Vitro and In Vivo. Oncology Research, 25(3), 365-372.

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Molecular Mechanisms of Ovarian Cancer

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Human ovarian cancer cell lines (SKOV-3, 2774 and OVCAR-3) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Human IFN-α and IFN-γ were obtained from Sigma (St. Louis, MO). The chemical doxazosin was also purchased from Sigma. The JAK1/2 kinase inhibitor INCB18424 (Ruxolitinib) and STAT1 inhibitor (NSC118218) were obtained from Selleck Chemicals (Houston, TX), and stock solutions were prepared in DMSO. NSC74859 (S31-201), a specific STAT3 inhibitor, was purchased from Calbiochem Chemicals (La Jolla, CA). The following primary antibodies were used in this study: anti-JAK1, anti-phospho-JAK1, anti-JAK2, anti-phospho-JAK2, anti-STAT1, anti-phospho-STAT1, anti-STAT3, anti-phospho-STAT3, anti-caspase-3, anti-cMyc, anti-Bcl-2, anti-Bax, anti-p53, anti-survivin, and anti-COX-2 (Cell Signaling, Beverly, MA), anti-PARP, anti-XIAP (BD Biosciences, San Jose, CA), anti-cyclin D1, anti-CDK4, anti-Akt, anti-phospho-Akt, anti-TYK2, anti-phospho-TYK2, anti-PI3K, anti-phospho-PI3K, anti-mTOR, anti-phospho-mTOR, anti-PKCδ, anti-phospho-PKCδ, anti-STAT2, anti-phospho-STAT2, anti-p70S6K, and anti-phospho-p70S6K (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p21, and anti-p27 (Oncogene, San Diego, CA).

Park M.S., Kim B.R., Kang S., Kim D.Y, & Rho S.B. (2014). The antihypertension drug doxazosin suppresses JAK/STATs phosphorylation and enhances the effects of IFN-α/γ-induced apoptosis. Genes & Cancer, 5(11-12), 470-479.

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Protein Extraction and Western Blot Analysis of Osteosarcoma Cells

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The proteins were extracted from osteosarcoma cells with RIPA buffer consisting of 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Equal amounts of protein were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk for 2 h and then incubated with primary antibodies (anti-HOXB7, anti-phospho-PI3K, anti-PI3K, anti-phospho-Akt, anti-Akt, or anti-GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. The blots were then washed and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence reagents (GE Healthcare, Buckinghamshire, UK). The signals were quantified by densitometry using Sion Image software (Scion Corporation, Frederick, MD, USA).

Yang L., Xie F, & Li S. (2017). Downregulation of Homeobox B7 Inhibits the Tumorigenesis and Progression of Osteosarcoma. Oncology Research, 25(7), 1089-1095.

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Western Blot Analysis of Renal Cell Carcinoma

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Proteins were extracted from RCC cells using cell lysis buffer, and protein concentration was determined using a Bradford protein assay (Takara Biotechnology, Dalian, P.R. China). Equal amounts of protein samples were loaded and isolated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto PVDF membranes (Millipore Corp., Billerica, MA, USA). After being blocked by 5% nonfat milk, the membranes were incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: anti-TPD52, anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-phospho-PI3K, anti-PI3K, anti-phospho-Akt, anti-Akt, and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, the film was incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. The immune-reactive protein bands were visualized by the ECL kit (Pierce, Rockford, IL, USA). Densitometric analysis was performed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).

Zhao Z., Liu H., Hou J., Li T., Du X., Zhao X., Xu W., Xu W, & Chang J. (2017). Tumor Protein D52 (TPD52) Inhibits Growth and Metastasis in Renal Cell Carcinoma Cells Through the PI3K/Akt Signaling Pathway. Oncology Research, 25(5), 773-779.

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Protein Expression Analysis by Western Blot

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Total protein extracts were lysed in lysis buffer [20 mM HEPES (pH 7.6), 350 mM NaCl, 20% glycerol, 0.5 mM EDTA, 0.1 mM EGTA, 1% NP-40, 50 mM NaF, 0.1 mM DTT, 0.1 mM PMSF, and a protease inhibitor cocktail]. The protein concentration was determined using a Bradford protein assay (Takara Biotechnology, Dalian, China). Equal amounts of protein were electrophoresed on SDS-PAGE and blotted onto PVDF membranes (Millipore Corp., Billerica, MA, USA). The membranes were blocked in 5% nonfat dry milk for 2 h and incubated with various primary antibodies (anti-SASH1, anti-E-cadherin, anti-N-cadherin, anti-phospho-PI3K, anti-PI3K, anti-phospho-Akt, anti-Akt or anti-β-actin; from Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C, followed by horseradish peroxidase (HRP)-conjugated IgG (Santa Cruz Biotechnology). The immunoreactive protein bands were visualized by ECL kit (Pierce, Rockford, IL, USA).

Zong W., Yu C., Wang P, & Dong L. (2016). Overexpression of SASH1 Inhibits TGF-β1-Induced EMT in Gastric Cancer Cells. Oncology Research, 24(1), 17-23.

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Comprehensive Western Blot Analysis of PI3K/AKT Pathway

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Western blot was performed as previously described 32 (link) with the following primary antibodies: anti-mTOR (1:100, sc-517464, Santa Cruz), anti-mTOR (1:1,000, #2972, Cell Signaling Technology), anti-phospho-IGF-1 Receptor (1:1000, #3021, Cell Signaling Technology), anti-phospho-HER-2(1:100, sc-81507, Santa Cruz), anti-Protor1 (1:100, sc-390496, Santa Cruz), anti-DEPTOR (1:100, sc-398169, Santa Cruz), anti-PDK1 (1:200, sc-293160, Santa Cruz), anti-PI3K p85α (1:100, sc-1673, Santa Cruz), anti-phospho-PI3K (1:100, sc-12929, Santa Cruz), anti-phospho-PI3K (1:1000, #4228, Cell Signaling Technology), anti-AKT (1:1,000, #4691T, Cell Signaling Technology), anti-phospho-AKT(S473) (1:1,000, #4060T, Cell Signaling Technology), anti-PHLPP2 (1:1,000, ab71973, Abcam), anti-phospho-AKT (T308) (1:100, sc-271966, Santa Cruz), anti-phospho-BAD (1:1000, #5284, Cell Signaling Technology), anti-phospho- GSK3β(Ser9) (1:1,000, #9323, Cell Signaling Technology), anti-phospho-FoxO1(Ser256) (1:1000, #9461T, Cell Signaling Technology), anti-phospho-4EBP1 (1:200, sc-293124, Santa Cruz), anti-phospho-p70S6K (1:200, sc-8416, Santa Cruz), anti-GAPDH (1:1,000, sc-47724, Santa Cruz), and anti-Myc-Tag (1:1,000, #2276s, Cell Signaling Technology).

Huang J., Li J., Tang J., Wu Y., Dai F., Yi Z., Wang Y., Li Y., Wu Y., Ren G, & Xiang T. (2022). ZDHHC22-mediated mTOR palmitoylation restrains breast cancer growth and endocrine therapy resistance. International Journal of Biological Sciences, 18(7), 2833-2850.

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Ovarian Cancer Cell Lines and HUVEC Culture

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Human ovarian cancer cell lines (SKOV-3 and OVCAR-3) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). They were grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 37°C in a humidified 5% CO₂ incubator. Primary HUVECs (Clonetics, San Diego, CA) were grown on 0.3% gelatin-coated dishes (Sigma-Aldrich, St. Louis, MO) in EGM-2 BulletKit medium (Clonetics). Rapamycin was purchased from Cell Signaling Technology (Beverly, MA). Doxazosin and all other chemicals were purchased from Sigma-Aldrich. The following primary antibodies were used: anti-phospho-VEGFR-2 (Y1175), anti-VEGFR-2, anti-phospho-PI3K, anti-PI3K, anti-phospho-Akt, anti-Akt, anti-HIF-1α, anti-phospho-ERK1/2, anti-ERK1/2 (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-PDK1, anti-PDK1, anti-phospho-4E-BP1, anti-4E-BP1, anti-phospho-mTOR, anti-mTOR, anti-phospho-p70S6K, anti-p70S6K, PCNA, cyclin D1, survivin (all from Cell Signaling Technology), anti-CD31 (Abcam, Cambridge, UK), anti-VEGF₁₆₅ (Ab-1; Oncogene, Cambridge, MA), and anti-β-actin (Sigma-Aldrich).

Park M.S., Kim B.R., Dong S.M., Lee S.H., Kim D.Y, & Rho S.B. (2014). The antihypertension drug doxazosin inhibits tumor growth and angiogenesis by decreasing VEGFR-2/Akt/mTOR signaling and VEGF and HIF-1α expression. Oncotarget, 5(13), 4935-4944.

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Comprehensive Western Blot Analysis

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Western blot was performed as previously described [27] with the following primary antibodies: anti-mTOR (1:100, sc-517464, Santa Cruz), anti-mTOR (1:1,000, #2972, Cell Signaling Technology), antiphospho-IGF-1 Receptor (1:1000, #3021, Cell Signaling Technology), anti-phospho-HER-2(1:100, sc-81507, Santa Cruz), anti-Protor1 (1:100, sc-390496, Santa Cruz), anti-DEPTOR (1:100, sc-398169, Santa Cruz), anti-PDK1 (1:200, sc-293160, Santa Cruz), anti-PI3K p85α (1:100, sc-1673, Santa Cruz), antiphospho-PI3K (1:100, sc-12929, Santa Cruz), anti-phospho-PI3K (1:1000, #4228, Cell Signaling Technology), anti-AKT (1:1,000, #4691T, Cell Signaling Technology), anti-phospho-Akt(S473) (1:1,000, #4060T, Cell Signaling Technology), anti-PHLPP2 (1:1,000, ab71973, Abcam), anti-phospho-Akt (T308) (1:100, sc-271966, Santa Cruz), anti-phospho-BAD (1:1000, #5284, Cell Signaling Technology), antiphospho-GSK3β(Ser9) (1:1,000, #9323, Cell Signaling Technology), anti-phospho-FoxO1(Ser256) (1:1000, #9461T, Cell Signaling Technology), anti-phospho-4EBP1 (1:200, sc-293124, Santa Cruz), antiphospho-p70S6K (1:200, sc-8416, Santa Cruz), anti-GAPDH (1:1,000, sc-47724, Santa Cruz), and antimyc-Tag (1:1,000, #2276s, Cell Signaling Technology).

Huang J., Li J., Tang J., Wu Y., Yi Z., Wang Y., Li Y., Tan Y., Ye L., Ren G., & Xiang T. (2021). ZDHHC22-Mediated mTOR Palmitoylation Determines Breast Tumor Growth and Endocrine Therapy Sensitivity.

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