Pkc θ sirna

Manufactured by Santa Cruz Biotechnology

Sourced in United States

PKC-θ siRNA is a synthetic short interfering RNA (siRNA) molecule designed to target and silence the expression of the PKC-θ (protein kinase C theta) gene. PKC-θ is a member of the protein kinase C family and plays a role in various cellular processes. The core function of PKC-θ siRNA is to facilitate the targeted knockdown of PKC-θ gene expression in experimental settings.

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4 protocols using pkc θ sirna

Modulating Cell Signaling in Breast Cancer

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MCF-7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM (Invitrogen, Life Technologies, Carlsbad, CA) supplemented with 10% FBS, 2mM L-glutamine, and 1% penicillin-streptomycin-neomycin. MCF-7 cells were stimulated with 1.32 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St Louis, MO) or 5 ng/ml recombinant TGF-β1 (R&D Systems, Minneapolis, MN) for 60 h, as previously described [28 ]. 0.65 nM triptolide (Santa Cruz, Dallas, TX) and 1 μM NSC 95397 (Santa Cruz) were used for inhibitor experiments. Forward transfection reactions were performed for 6 h with 50 nM human DUSP1 siRNA (sc-35937), DUSP4 siRNA (sc-38998), DUSP6 siRNA (sc-39000), 20 nM PKC-θ siRNA (sc-36252), and 10 nM MOCK siRNA (sc-36869) (Santa Cruz) using Lipofectamine 2000 (Invitrogen).

Boulding T., Wu F., McCuaig R., Dunn J., Sutton C.R., Hardy K., Tu W., Bullman A., Yip D., Dahlstrom J.E, & Rao S. (2016). Differential Roles for DUSP Family Members in Epithelial-to-Mesenchymal Transition and Cancer Stem Cell Regulation in Breast Cancer. PLoS ONE, 11(2), e0148065.

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Immunoblot Analysis of PKC-θ Expression

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Immunoblot analysis was performed using primary mouse antibody to human SC35p (as above, ab11826) and secondary HRP-conjugated goat-anti-mouse antibody on nuclear extracts isolated from either primary human CD4⁺ cells, which were untreated (mock) or treated with Life Technologies siRNA PKC-θ pool (Life Technologies ID s11122, s11123), PKC-θ siRNA (Santa Cruz SC-36252), or nuclear extracts from NS or ST Jurkat T cells either untreated or treated with rottlerin. Nuclear extracts of transfected Jurkat T cells were also probed for nuclear expression of HA-tagged PKC-θ WT or NLS mutant constructs using a primary rabbit antibody to HA (Sigma, H6908). Signals were detected with enhanced chemiluminescence reagents (Western Lightning ECL-Plus, Perkin-Elmer NEL104001) and film exposure. Band intensity signals were normalized to the total protein transferred to the blot detected using the Quantitative Novex Reversible Protein Stain (Thermo Fisher Scientific IB7710) and Image J analysis.

McCuaig R.D., Dunn J., Li J., Masch A., Knaute T., Schutkowski M., Zerweck J, & Rao S. (2015). PKC-Theta is a Novel SC35 Splicing Factor Regulator in Response to T Cell Activation. Frontiers in Immunology, 6, 562.

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Insulin Signaling in 3T3-L1 Adipocytes

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Mouse 3T3-L1 preadipocytes were obtained from American Type Culture Collection (Manassas, VA). 3T3-L1 preadipocyte medium, 3T3-L1 adipocyte differentiation medium, and 3T3-L1 adipocyte maintenance medium were obtained from Zen-Bio, Inc. (Research Triangle Park, NC). [1, 2-³H] 2-deoxy-D-glucose was purchased from Perkin Elmer Life Sciences (Waltham, MA). Hypochlorous sodium solution, human insulin, Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), SP600125, and antibody against phospho-IRS-1 (Tyr612) were purchased from Sigma-Aldrich (St. Louis, MO). Protein A/G-agarose, RIPA lysis buffer, PS-1145, IKKβ siRNA, JNK2 siRNA, PKCθ siRNA, control siRNA, and antibodies against β-actin, GAPDH and Na⁺/K⁺ ATPase were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phospho-IRS-1 (Ser307), IRS-1, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, phospho-GSK3β (Ser9), GSK3β, phospho-IKKα/β (Ser176/180), IKKα, IKKβ, phospho-SAPK/JNK (Thr183/Tyr185), JNK, phospho-PKCθ (Thr538), PKCθ, GLUT4, IκBα, and horseradish peroxidase-linked secondary antibodies (Beverly, MA). ONOO⁻ was obtained from Calbiochem (Billerica, MA). All other chemicals were of the highest commercial grade available.

Zhou J., Wang Q., Ding Y, & Zou M.H. (2015). Hypochlorous acid via peroxynitrite activates protein kinase Cθ and insulin resistance in adipocytes. Journal of Molecular Endocrinology, 54(1), 25-37.

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Insulin Resistance in 3T3-L1 Adipocytes

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Mouse 3T3-L1 preadipocytes were obtained from American Type Culture Collection (Manassas, VA, USA). 3T3-L1 preadipocyte medium, 3T3-L1 adipocyte differentiation medium, and 3T3-L1 adipocyte maintenance medium were obtained from Zen-Bio, Inc. (Research Triangle Park, NC, USA). [1,2-³H] 2-deoxy-d-glucose was purchased from Perkin Elmer Life Sciences (Waltham, MA, USA). Hypochlorous sodium solution, human insulin, Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), SP600125, and antibody against phospho-IRS1 (Tyr612) were purchased from Sigma–Aldrich. Protein A/G-agarose, RIPA lysis buffer, PS-1145, IKKβ siRNA, JNK2 siRNA, PKCθ siRNA, control siRNA, and antibodies against β-actin, GAPDH, and Na⁺/K⁺ ATPase were obtained from Santa Cruz Biotechnology, Inc. Antibodies against phospho-IRS1 (Ser307), IRS1, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, phospho-GSK3β (Ser9), GSK3β, phospho-IKKα/β (Ser176/180), IKKα, IKKβ, phospho-SAPK/JNK (Thr183/Tyr185), JNK, phospho-PKCθ (Thr538), PKCθ, GLUT4, IκBα, and HRP-linked secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). ONOO⁻ was obtained from Calbiochem (Billerica, MA, USA). All other chemicals were of the highest commercial grade available.

Zhou J., Wang Q., Ding Y, & Zou M.H. (2015). Hypochlorous acid via peroxynitrite activates protein kinase Cθ and insulin resistance in adipocytes. Journal of Molecular Endocrinology, 54(1), 25-37.

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