Whole-cell lysates of transfected human embryonic kidney (HEK) 293T cells, neutrophils, and stimulated MDMs were prepared in a modified radioimmunoprecipitation assay buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40 and 1 mM EDTA) supplemented with protease inhibitor cocktail (#5,892,970,001, Merck) and phosphatase inhibitor cocktail (#4,906,837,001, Merck), 0.1 mM dithiothreitol (DTT; #20,290, Thermo Fisher Scientific), and 1 mM PMSF (#10,837,091,001, Merck). Protein concentrations were determined by Bradford protein assay, and 30 µg of protein was subjected to SDS-PAGE in Criterion TGX 10% precast gels (#5,671,033, BIO-RAD). The resulting bands were transferred onto nitrocellulose membranes (#1,704,159, BIO-RAD) with the Trans-Blot Turbo Transfer System (#1,704,150, BIO-RAD). Membranes were blocked by incubation in 3% BSA in PBS-T and were then incubated with antibodies directed against PKCδ, p-PKCδ (T505; #9374, Cell Signaling Technology), p-PKCδ (S643; #9376, Cell Signaling Technology), p40phox (#07–503, Merck), p-p40phox (T154; #4311, Cell Signaling Technology), vinculin (#sc-73614, Santa Cruz), and GAPDH (#sc-47724, Santa Cruz). Proteins were visualized with the Clarity Western ECL substrate (#1,705,061, BIO-RAD) or SuperSignal West Femto (#34,096, Thermo Fisher Scientific), with ChemiDoc MP (Biorad). Images were analyzed with Imagine Lab 5.1 (Bio-Rad Laboratories).
P pkcδ
Manufactured by Cell Signaling Technology
Sourced in United States
P-PKCδ is an antibody that detects the phosphorylated form of protein kinase C delta (PKCδ). PKCδ is a serine/threonine protein kinase that plays a role in various cellular processes, including cell proliferation, differentiation, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
P erk, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
P pkc α β 2, by Cell Signaling Technology (2 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Criterion tgx 10 precast gels, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Vinculin, by Santa Cruz Biotechnology (1 mentions)
P p40phox, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P jak, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
10 protocols using p pkcδ
1
Analysis of Phosphorylated Proteins in Cell Lysates
2
Ingenol Mebutate Regulation of Apoptosis
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Ingenol mebutate was the product of Nanjing Spring and Autumn Biological Engineering Co., Ltd., Nanjing, China. Antibodies against survivin, AKT, p-AKT, ERK, p-ERK, p-PKCδ, PKCδ, p-JAK, JAK, p-STAT3, and STAT3, were purchased from Cell Signaling Technology, Beverly, MA, USA. Annexin V-FITC/PI apoptosis detection kit was provided by Nanjing KeyGEN BioTECH. Co., Ltd., Nanjing, China. Other reagents and kits were the products of Beyond, Nantong, China.
3
Western Blot Analysis of Stress Proteins
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Western blot analyzes were performed as described previously16 (link) using primary antibodies targeting the following proteins: PLOD3 (Proteintech Group, Chicago, IL, USA); p-PERK, PERK, p-eIF2α, HA, GFP, lamin A/C, α-tubulin, PKCα, PKCδ (Santa Cruz Biotechnology); cleaved-PARP (Asp214), cleaved caspase-3, eIF2α, IRE1α, γH2AX, and p-PKCδ (Cell Signaling Technology); p-IRE1α (Novous); ATF6, caspase-2 and caspase-4 (Abcam); and β-actin (Sigma) was used as a loading control.
4
Western Blot Analysis of Signaling Pathways
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Cells were lysed in a sample buffer containing 2% SDS, 60 mmol/L Tris‐HCl (pH 6.8) and 5% glycerol. Cell lysates were boiled for 5 minutes. Protein concentration was determined using a BCA kit (Beyotime, Shanghai, China), and equal amount of protein was loaded for Western blot analysis as previously described.11 Primary antibodies against ERK1/2, p‐ERK1/2, p38, p‐p38, JNK1/2, p‐JNK1/2, PKCδ, p‐PKCδ, PKA, p‐PKA, MARK2, p‐MAPK, LKB1, p65 and p‐p65, as well as secondary antibodies, were all from Cell Signaling Technology (Boston, MA, USA). Anti‐β‐actin antibody was from Proteintech (Wuhan, China). Blots were developed using enhanced ECL chemiluminescence reagents (Pierce, Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instruction. β‐actin was used as a loading control.
5
Western Blot Analysis of Liver and RAW 264.7 Cells
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Liver tissues and RAW 264.7 cells were homogenized in NP40 buffer containing 50 mM HEPES pH 7.4, 150 mM NaCl, 1% NP40, 1 mM EDTA, and protease inhibitor cocktail tablets (Roche, Mannheim, Germany). After electroblotting, the proteins were separated onto nitrocellulose membranes (GE Healthcare Life science), the membranes were blocked with 5% w/v skim milk in TBST (50-mM Tris pH 8.0, 150-mM NaCl, and 0.1% Tween-20 (v/v)) for 1 h. The membranes were immunoblotted with antibodies against α-SMA (Sigma-Aldrich, St Louis, MO, USA), acetyl-lysine (Merck Millipore KGaA, Darmstadt, Germany), p-PKCα, p-PKCδ, p-PKC ζ, P65, SIRT1, iNOS (Cell signaling; Danvers, MA, USA), PKCα, PKCδ, and β-actin (Santa Cruz, CA, USA). β-actin was used as the loading control. Primary antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies. Membranes were washed in TBST and then probed with the appropriate horseradish peroxidase-coupled secondary antibody (Cell Signaling).
6
Measuring Oxidative Stress Signaling
7
Investigating Protein Signaling in Cell Stress
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Cells (3.0 × 107) were treated with 0.5 mM NAC, BA (12.5, 25, 50 or 100 μg/ml) and an equal volume of RPMI-1640 medium for 2 h. HPS (3.0 × 107 CFU/ml) was added and the protein was extracted after 6 h using RIPA Lysis Buffer Strong (P0013, Beyotime Biotechnology, Shanghai, China). The protein concentration was determined by BCA protein assay kit (P0012, Beyotime Biotechnology). The proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (60 mA, 1 h). The PVDF membrane was blocked with 5% skimmed milk at 20 °C for 3 h, then incubated with the primary antibody (containing 5% BSA TBS-T solution, 1:1000 dilution, Anti β-actin, cleaved caspase-3, JNK, p-JNK, p38, p-p38, ERK, p-ERK, PKC-α and p-PKC-δ, Cell Signaling Technology, MA, USA; and p-PKC-α and PKC-δ, Sigma, USA) overnight at 4 °C and the secondary antibody (containing 5% skimmed milk TBS-T solution, 1:2000 dilution; Cell Signaling Technology) at room temperature for 3 h. Afterwards, the membrane was visualized by ECL solution (Thermo Pierce ECL, USA) using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). The β-actin was used as an inner loading control. Gray value was analyzed and the relative expression level of protein was obtained.
8
HPLC Analysis of Bioactive Compounds
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Standard samples (chlorogenic acid and γ-aminobutyric acid (GABA)) for high-performance liquid chromatography (HPLC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution, lipopolysaccharide (LPS), vitamin C, and dexamethasone were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Recombinant human interferon (IFN)-γ and tumor necrosis factor (TNF)-α were obtained from BioLegend (San Diego, CA, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Gibco BRL (Rockville, MD, USA). Enzyme-linked immunosorbent assay (ELISA) kit for histamine was obtained from Abcam (Cambridge, UK) for histamine. Antibodies against p-p38, p38, p-ERK, ERK, p-JNK, JNK, p-Lyn, Lyn, p-Syk, Syk, p-Fyn, Fyn, p-PLCγ1, PLCγ1, p-PKCδ, PKCδ, NF-κB p65, Lamin B, Nrf2, HO-1, NQO1, iNOS, COX-2, and β-actin were purchased from Cell Signaling Technology (Danvers, MN, USA) or Santa Cruz (Dallas, TX, USA).
9
Ginsenoside CK Modulates Inflammatory Pathways
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Black ginseng extracts were prepared, and ginsenoside compound K (CK, purity >95%) was provided by Dr. Dae Young Lee (Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Soi-myeon, Eumseong-gun, Chungbuk, Republic of Korea). Research cigarette 2R4F was obtained from the Tobacco and Health Research Institute (University of Kentucky, Lexington, KY). PMA, LPS, DMSO (Dimethyl Sulfoxide), roflumilast (ROF), and rottlerin were obtained from Sigma-Aldrich (St. Louis, MO). Anti-phospho(p)–NF–κB, –NF–κB, -p-p70S6K, -p70S6K, -p-mTOR, -mTOR, -EGR-1, -p-RSK, -RSK, -p-CREB, -CREB, and -p-PKCδ antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-p-JNK, -JNK, -p-ERK, -ERK, -PKCδ, and -β-actin antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA).
10
Euphol-induced Protein Expression Analysis
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To evaluate the expression of altered proteins following euphol treatment, the cells were plated onto a six-well plate at a density of 1 × 10 6 cells/well, allowed to adhere at least 24 h, and were serum-starved in DMEM (0.5% FBS). The cells were exposed at IC 50 concentration values of euphol for an additional period of 6, 24, 48 and 72 h in DMEM (0.5% FBS). After the cells were prepared and aliquots of 20 μg of total protein were separated as previously described for western-blot analysis [27] (link). Antibodies included anti-Bip, Rip, p-27, DR5, SOD2, CytC, pP53 (S15), HSP60, HSP70, FADD and β-tubulin, all antibodies were diluted at 1:1000, (Cell Signalling Technology). In addition to PKC profile analysis, antibodies included total PKCs (PKCα, PKCδ and PKCζ), and phosphorylated PKCs; p-PKC PKDμ (S916), p-PKC PKDμ (S744), p-PKCα/βII, p-PKCpanβII, p-PKCδ, p-PKCδ/θ, p-PKCθ and PKCζ/λ, all antibodies were diluted at 1:1000 and purchased from Cell Signalling Technology.
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