Pikbαs32

Manufactured by Cell Signaling Technology

PIKBαS32 is a recombinant protein that contains the serine 32 phosphorylation site of the PIKBα (Phosphoinositide-3-Kinase Regulatory Subunit Alpha) protein. It is used as a tool in research to study the phosphorylation of the PIKBα subunit.

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Lab products found in correlation

3 protocols using pikbαs32

Interrogating Cellular Signaling Pathways in Colorectal Cancer

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Approximately, 30 to 80 mg of nuclear or cytoplasmic protein that was isolated from SW480 colorectal cancer cells using NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, Waltham, MA) was loaded and separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with the following antibodies overnight at indicated dilutions. pNFκB P65 ^S536, 1:1000, NFκB (P65), 1:1000, pGSK-3α^S21β^S9, 1:1000, pGSK3α^Y51β^Y47, 1:2000, GSK-3β, 1:1000, COX-2, 1:1000, pIKBα^S32, 1:1000, IKBα, 1:1000, pIKKα/IKKβ^S176/180, 1:1000, IKKα/IKKβ, 1:1000, pAkt^S473, 1:2000, Akt, 1:2000, β-Catenin, 1:1000, pβ-Catenin^S33/37/T41, 1:1000 (Cell Signaling Technology, Danvers, MA); Each membrane was probed with P84 (Gene Tex, Irvine, CA) to ensure consistent loading of nuclear protein and β-actin, 1:5000 (Sigma, St. Louis, MO) to ensure consistent loading of cytoplasmic protein.

Saud S.M., Li W., Gray Z., Matter M.S., Colburn N.H., Young M.R, & Kim Y.S. (2016). Diallyl Disulfide (DADS), a constituent of garlic, inactivates NFκB and prevents colitis-induced colorectal cancer by inhibiting GSK-3β. Cancer prevention research (Philadelphia, Pa.), 9(7), 607-615.

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Immunoblot and Immunoprecipitation of Key Signaling Proteins

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Immunoblot and immunoprecipitation were performed according to the standard procedures (Kim et al., 2018 (link)). The following primary antibodies were used: anti-HDAC6, TLR2, TLR4, and SIRT1 (ABclonal); anti-FcεRIβ, Lyn, GATA3, T-bet, JNK1, pJNK1^T183/Y185, Tryptase, Chymase, BECN1, MyD88, TSG101, and Calnexin (Santa Cruz Biotechnology); anti-CXCL13 (R&D Systems); anti-CD163, FoxP3, TSLP, and MIP-2 (Abcam); anti-iNOS, pBECN1^S14, COX2, ERK1/2, pERK^T204, HDAC3, NFκB, AMPKα, pAMPKα^T172, IKBα, pIKBα^S32, p38MAPK, p-p38MAPK^T180/Y182, and LC3(Cell Signaling Technology). The detailed information of primary antibodies is described in Supplemental Table S2.To isolate tissue lysates, tissue was frozen in liquid nitrogen to preserve protein structure and homogenized with RIPA buffer. After lysis, vortexing and centrifugation at 10,000 X g for 15 min at 4°C were followed. Supernatant was then obtained and used as tissue lysates for immunoblot and immunoprecipitation.

Kwon Y., Choi Y., Kim M., Jeong M.S., Jung H.S, & Jeoung D. (2021). HDAC6 and CXCL13 Mediate Atopic Dermatitis by Regulating Cellular Interactions and Expression Levels of miR-9 and SIRT1. Frontiers in Pharmacology, 12, 691279.

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Probing the mTOR Signaling Pathway

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Whole-cell extracts were prepared using 2X SDS buffer supplemented with fresh protease and phosphatase inhibitors. Equal volumes of protein were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The following antibodies were used: Akt, mTOR, p-mTOR^S2448, p-mTOR^S2481, ribosomal protein S6, phospho-ribosomal protein S6^S235/236, Rictor, Raptor, TSC2, p-TSC2^S939, NFκB p50, NFκB p65, IκBα, p-IkBα^S32, and IKKα (purchased from Cell Signaling Technology, Danvers, MA). β-actin antibody was purchased from Sigma-Aldrich. Anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Blots were developed using Western Lighting Plus ECL chemiluminescent reagents (Perkin Elmer, Waltham, MA) and Syngene G:Box Imaging System (Frederick, MD). Band quantification was performed using Syngene Genetools gel analysis software and band intensities normalized to β-actin intensity.

Hambright H.G., Batth I.S., Xie J., Ghosh R, & Kumar A.P. (2014). Palmatine inhibits growth and invasion in prostate cancer cell: potential role for rpS6/NFκB/FLIP. Molecular carcinogenesis, 54(10), 1227-1234.

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