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Phospho pkcδ

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Phospho-PKCδ is a primary antibody product offered by Cell Signaling Technology. It is designed to detect the phosphorylated form of protein kinase C delta (PKCδ), a serine/threonine protein kinase that plays a role in various cellular processes.

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Lab products found in correlation

Phospho p38, by Cell Signaling Technology (3 mentions) Bace1, by Cell Signaling Technology (2 mentions) Phospho syk, by Cell Signaling Technology (2 mentions) Phospho lyn, by Cell Signaling Technology (2 mentions) Cox 2, by Cell Signaling Technology (2 mentions) Phospho jnk, by Cell Signaling Technology (2 mentions) Phospho erk, by Cell Signaling Technology (2 mentions) Dulbecco phosphate buffered saline (dpbs), by GE Healthcare (2 mentions) Specific antibody against phospho fyn, by Biorbyt (2 mentions) Mem α medium, by GE Healthcare (2 mentions) Ez cytox cell viability assay kit, by Daeil Lab Service (2 mentions) Fetal bovine serum (fbs), by GE Healthcare (2 mentions) Wortmannin, by Cayman Chemical (1 mentions) Ldh assay, by Promega (1 mentions) Rottlerin, by Cayman Chemical (1 mentions) Bisindolylmaleimide 1, by Cayman Chemical (1 mentions) Phospho pi3k, by Cell Signaling Technology (1 mentions) Pi3k p85, by Cell Signaling Technology (1 mentions) G 6983, by Cayman Chemical (1 mentions) Ly294002, by Cayman Chemical (1 mentions)

12 protocols using phospho pkcδ

1

Integrin-mediated Signaling Pathway Analysis

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All reagents unless otherwise specified were from Sigma-Aldrich. Wortmannin, Ly294002, rottlerin, bisindolylmaleimide i, bisindolylmaleimide ix, Gö6983 were from Cayman Chemical. P11, GRGDSP, GRADSP, αvβ3 function blocking antibody (LM609) were from EMD Millipore. PP1, PP2, PP3 were from Calbiochem. LDH assay was from Promega. Phospho-MARCKS Ser152/156 (Cat#2741), MARCKS (Cat#5607), Phospho-PI3K (Cat#4228), p85 PI3K (Cat#4257), Phospho-PKCδ (Cat#2055), PKCd (Cat#9616) antibodies were from Cell Signaling Technology. FITC-PIP3 antibody (Cat#G345) was from Echelon. All secondary antibodies and isotype controls were from Life Technologies.
Cornick S., Moreau F, & Chadee K. (2016). Entamoeba histolytica Cysteine Proteinase 5 Evokes Mucin Exocytosis from Colonic Goblet Cells via αvβ3 Integrin. PLoS Pathogens, 12(4), e1005579.
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2

Cardiac Protein Extraction and Analysis

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Whole-heart protein lysates made from freeze-clamped hearts using lysis buffer (Hepes pH 7.4 (20mM), β-Glycerol phosphate (50 mM), EGTA (2mM), DTT (1mM), NaF (10mM), NaVO4 (1mM), Triton-X 100 (1%), Glycerol (10%,) and 1 protease inhibitor complete mini tablet-EDTA free/20 ml (Roche)) were subjected to SDS-PAGE with protein content quantified using the Bradford method (Biorad). Following transfer, nitrocellulose membranes were probed with primary antibodies to MFN1 (Neuromab), MFN2 (Sigma-Aldrich AV42420), OPA1 (BD Transduction 612606), phospho-PKCpan (Cell Signaling 9371), phospho-PKC α (Cell Signaling 06-822), total PKC α (EMD 05-154), phospho-PKC δ (Cell Signaling 9376), total PKC δ (Santa Cruz 937), phospho-PKC ε (Santa Cruz sc12355), total PKC ε (EMD 06-991), SDHB (Abcam ab110413), CREB (Pierce MA1-083), GAPDH (Abcam ab9485), and visualized using horseradish peroxidase secondary antibodies with SuperSignal West chemiluinescence substrate and an enhanced chemiluminescence Fujifilm LAS-4000 imager or LI-COR secondary antibodies and the LICOR Odyssey IR imager. Densitometry was quantified using Fujifilm Multigauge or Odyssey software.
Elezaby A., Sverdlov A.L., Tu V.H., Soni K., Luptak I., Qin F., Liesa M., Shirihai O.S., Rimer J., Schaffer J.E., Colucci W.S, & Miller E.J. (2014). Mitochondrial Remodeling in Mice with Cardiomyocyte-Specific Lipid Overload. Journal of molecular and cellular cardiology, 79, 275-283.
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3

Immunofluorescence Analysis of Phospho-Kinases

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HR1R2F cells cultured in 96-well plates were starved in serum-free DMEM for 16 hrs, followed by treatment with 0.5 μM fMLF for 20 min. Cells were fixed and the immunofluorescence was assessed following staining with Becton Dickinson Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit following the manufacturer’s protocol. Phospho-PKD/PKμ (Ser 744/748), phospho-PKCα/β II (Thr638/641), phospho-PKCδ (Thr505); phospho-PKC θ (Thr538), phospho-PKC ζ/λ (Thr410/403) (Cell signaling technology Inc.) were prepared in the BD washing buffer and added to the cells at 4°C, then stained with alexa-488 labeled secondary antibodies for 2 hrs at 4°C, and imaged with a Nikon inverted fluorescence microscope using a 20× objective lens.
Bednar F., Song C., Bardi G., Cornwell W, & Rogers T.J. (2014). Cross-Desensitization of CCR1, But Not CCR2, Following Activation of the Formyl Peptide Receptor (FPR1). Journal of immunology (Baltimore, Md. : 1950), 192(11), 5305-5313.
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4

BACE1 Inhibition in Cell Signaling

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Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). A Cell Counting Kit-8 (CCK8) was purchased from Beyotime (Shanghai, China), β-secretase inhibitor IV (BACE1 inh.) was purchased from Biochemicals (Solon, OH, USA), and phorbol-12-myristate-13-acetate (PMA) was purchased from Sigma (St. Louis, MO, USA). Polyclonal antibodies against phospho-MEK, phospho-PKCδ, phospho-ERK, BACE1, MEK, PKCδ, and ERK and the monoclonal antibody against β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA), and the anti-ST6GAL1 antibody was purchased from Santa (Dallas, Texas, USA).
Deng X., Zhang J., Liu Y., Chen L, & Yu C. (2017). TNF-α regulates the proteolytic degradation of ST6Gal-1 and endothelial cell-cell junctions through upregulating expression of BACE1. Scientific Reports, 7, 40256.
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5

Investigating Kinase Signaling in Cells

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Cells were treated with various doses of MK-2206 (Selleck Chemicals) for 6 h or 5 μM GSK3 Inhibitor XVI (Calbiochem) for 6 h. Cells were sonicated in lysis buffer (62.5 mM Tris (pH 8.0), 2% SDS, 10% glycerol, 100 μM AEBSF, 80 nM aprotinin, 5 μM bestatin, 1.5 μM E-64, 2 μM leupeptin, 1 μM pepstatin, 500 μM sodium orthovanadate, 500 μM glycerol phosphate, 500 μM sodium pyrophosphate and 50 μM DTT) and protein (5 × 105 cell equivalents) was subjected to electrophoresis using 10–14% acrylamide/0.1% SDS gels. Proteins were transferred to a nitrocellulose membrane and Western blotting analysis was performed with antibodies against phospho-PKCδ (Cell Signaling, Beverly, MA), PKCδ (Santa Cruz Biotechnology), phospho-GSK3α/β (serine 9/21; Cell Signaling), GSK3α/β (Cell Signaling), MYC (Cell Signaling), Foxo3A (Cell Signaling), and Tubulin (Sigma). Signals were detected by using the Odyssey Infrared Imaging System and quantitated by Odyssey software version 3.0 (both LI-COR Biosciences, Lincoln, NE, USA). Tubulin was used as a loading control.
Ruvolo P.P., Qiu Y., Coombes K.R., Zhang N., Neeley E.S., Ruvolo V.R., Hail N J.r., Borthakur G., Konopleva M., Andreeff M, & Kornblau S.M. (2015). Phosphorylation of GSK3α/β correlates with activation of AKT and is prognostic for poor overall survival in acute myeloid leukemia patients. BBA Clinical, 4, 59-68.
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6

Quantitative Immunoblotting Analysis of Signaling Proteins

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Cells were lysed in 50 mM HEPES (pH 7.5), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 2 mM EDTA, and 10 mM NaF with protease inhibitors (Roche) and phosphatase inhibitors (Calbiochem). Lysates were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes using the iBlot system (Life Technologies). Two-color immunoblotting was performed using LI-COR reagents (Odyssey Blocking Buffer and IRDye 800CW and IRDye 680RD secondary antibodies) according to the manufacturer’s instructions (LI-COR Biosciences). Fluorescence detection was performed using an Odyssey CLx Infrared Imaging System, and quantitation was performed using Image Studio software (LI-COR). Antibodies against ALK (#3791), AKT (#2920), phospho-AKT (Ser 473; #4060), EGFR (#2239), phospho-EGFR (Tyr 1068; #3777), PKCδ (#9616), and phospho-PKCδ (Tyr 311; #2055) were obtained from Cell Signaling. ERK2 antibody (sc-154) was purchased from Santa Cruz Biotechnology. Antibodies against phospho-ERK1/ERK2 (Thr 185/Tyr 187; #M8159) and vinculin (#V9131) were obtained from Sigma. V5 antibody (#R960-25) was obtained from Life Technologies. In-cell Western blotting is described in the Supplemental Experimental Procedures.
Wilson F.H., Johannessen C.M., Piccioni F., Tamayo P., Kim J.W., Van Allen E.M., Corsello S.M., Capelletti M., Calles A., Butaney M., Sharifnia T., Gabriel S.B., Mesirov J.P., Hahn W.C., Engelman J.A., Meyerson M., Root D.E., Jänne P.A, & Garraway L.A. (2015). A Functional Landscape of Resistance to ALK Inhibition in Lung Cancer. Cancer cell, 27(3), 397-408.
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7

Immunoblotting of Retinal Proteins

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Whole retinal lysates and REC lysates were placed into lysis buffer containing protease and phosphatase inhibitors. The lysates were kept on ice for 30 min and cleared by centrifugation at 12,000 rpm for 20 min at 4 °C. Equal amounts of protein from the lysates were separated on the precast tris-glycine gels (Invitrogen, Carlsbad, CA) and blotted onto a nitrocellulose membrane. The blots were blocked with TBST (10mM Tris-HCl buffer, pH 8.0, 150mM NaCl, 0.1% Tween 20) containing 5% (w/v) bovine serum albumin and then incubated with respective primary antibodies. Primary antibodies used were anti-phospho-PKCζ, PKCζ, PKCδ, phospho-PKCδ, phospho-eNOS and eNOS (Cell Signaling, Danvers, MA). IGFBP-3, VEGF and β-actin antibodies were purchased from Santa Cruz Technology (Santa Cruz, CA). Membranes were then washed with TBST and incubated with horseradish peroxidase labeled secondary antibodies. The antigen–antibody complexes were detected using West Pico Chemilluminescence reagent. Mean densitometry of the bands were assessed using Kodak Image Station 4000MM software (Carestream, Rochester, NY).
Jiang Y., Zhang Q, & Steinle J.J. (2015). Beta-adrenergic receptor agonist decreases VEGF levels through altered eNOS and PKC signaling in diabetic retina. Growth factors (Chur, Switzerland), 33(3), 192-199.
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8

Cell Viability and Inflammatory Mediator Analysis

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DMEM, MEM-α medium, 1 × DPBS, antibiotics, and fetal bovine serum (FBS) were purchased from GE Healthcare Life Sciences (Hyclone™, Logan, UT, USA). The EZ-Cytox cell viability assay kit was obtained from Daeil Lab Service Co., Ltd. (Seoul, Korea). Specific antibodies against phospho-Akt, phospho-cPLA2, phospho-ERK, phospho-JNK, phospho-Lyn, phospho-p38, phospho-PKCδ, phospho-PLCγ1/2, phospho-Syk, and COX-2 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). A specific antibody against phospho-Fyn was obtained from Biorbyt Ltd. (Cambridge, UK). A specific antibody against β-actin was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). A specific antibody against phospho-5-LO and EIA kits for PGD2 and LTC4 were obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). ELISA kits for IL-4 and TNF-α were purchased from e-Bioscience, Inc. (Science Center Drive, San Diego, CA, USA). A specific antibody against MMP-1 was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Specific antibodies against MMP-3 and MMP-13 were purchased from Abcam, Inc. (Cambridge, UK). 4-Nitrophenyl N-acetyl-β-d-glucosaminide (p-NAG), casein, DNP-HSA, DNP-IgE, trichloroacetic acid, caffeic acid, diethylene glycol, Folin-Ciocalteu reagent, and quercetin were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.
Yoo J.M., Yang J.H., Kim Y.S., Yang H.J., Cho W.K, & Ma J.Y. (2016). Inhibitory Effects of Viscum coloratum Extract on IgE/Antigen-Activated Mast Cells and Mast Cell-Derived Inflammatory Mediator-Activated Chondrocytes. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(1), 37.
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9

Cell Viability and Inflammatory Mediator Assays

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MEM-α medium, 1 × DPBS, penicillin, streptomycin and fetal bovine serum (FBS) were purchased from GE Healthcare Life Sciences (Hyclone™, Logan, UT, USA). The EZ-Cytox cell viability assay kit was obtained from Daeillab Service Co. (Seoul, Korea). Specific antibodies against phospho-cPLA2, phospho-ERK, phospho-JNK, phospho-LYN, phospho-p38, phospho-PKCδ, phospho-PLCγ1, phospho-SYK and COX-2 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). A specific antibody against phospho-FYN was obtained from Biorbyt Ltd. (Cambridge, UK). A specific antibody against α-tubulin was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). An enzyme immunoassay (EIA) kit for PGD2 was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-4, and IL-6 were purchased from e-Bioscience, Inc. (San Diego, CA, USA). 4-Nitrophenyl-N-acetyl-β-d-glucosaminide (p-NAG), dinitrophenyl-human serum albumin (DNP-HSA) and DNP-immunoglobulin E (DNP-IgE) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.
Park K.I., Kim D.G., Yoo J.M, & Ma J.Y. (2016). The Herbal Medicine KIOM-MA128 Inhibits the Antigen/IgE-Mediated Allergic Response in Vitro and in Vivo. Molecules, 21(8), 1015.
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10

Western Blot Analysis of Lipid Metabolism Proteins

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Liver samples were quickly-frozen in liquid nitrogen and stored at −80 °C. Procedures of protein extraction and western blot were carried out as described previously [36 (link)]. Polyclonal goat antibody against DGAT1 (Sigma-Aldrich Inc., St. Louis, MO, USA), polyclonal mice antibody against monoclonal rabbit antibody against ATGL and polyclonal rabbit antibody against ACSL1 (Abcam Inc., Cambridge, MA, USA) and polyclonal rabbit antibodies against IRS1, Phospho-IRS1 (Ser302), PI3Knase (PI3K), Phospho-PI3Knase p85 (Tyr458)/p55 (Tyr199), AKT, Phospho-AKT(Ser473), PKCδ, Phospho-PKCδ (Ser359) (Cell Signaling Technology Inc., Danvers, MA, USA); Horseradish peroxidase conjugated secondary antibody (KANGCHEN Biotechnology, Nanjing, China) were examined by standard western blotting protocol. Respective proteins were analyzed with GAPDH serving as the loading control.
Zhao X., Shen C., Zhu H., Wang C., Liu X., Sun X., Han S., Wang P., Dong Z., Ma X., Hu K., Sun A, & Ge J. (2016). Trans-Fatty Acids Aggravate Obesity, Insulin Resistance and Hepatic Steatosis in C57BL/6 Mice, Possibly by Suppressing the IRS1 Dependent Pathway. Molecules, 21(6), 705.
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