Western blot analysis was performed as described previously [33 (link)] with minor modifications. Briefly, aliquots of 30–60 µg of protein were subjected to 8–12% SDS-PAGE and transferred onto Hybond-P+ polyvinylidene difluoride membranes (Amersham, Buckinghamshire, UK). The membranes were incubated with the following primary antibodies: anti-ICAM-1 (ab225884, 1:1000, Abcam, Cambridge, UK), anti-VCAM-1 (ab106778, 1:1000, Abcam), anti-phospho-ERK (sc-7383, 1:1000, Santa Cruz Biotechnology), anti-total-ERK (sc-94 1:1000, Santa Cruz Biotechnology), anti-phospho-PKC (9375S, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-PKC (sc-10800, 1:1000, Santa Cruz Biotechnology), anti-phospho-PDK1 (3061S, 1:1000, Cell Signaling Technology), anti-PDK1 (3062S, 1:1000, Cell Signaling Technology), anti-phospho-STAT-3 (9131S, 1:1000, Cell Signaling Technology, anti-STAT-3 (4904S, 1:1000, Cell Signaling Technology), anti-HIF-1α (ab2185, 1:1000, Abcam), anti-phospho-NF-κB (8242S, 1:1000, Cell Signaling Technology), anti-NF-κB (8242S 1:1000, Cell Signaling Technology), and anti-β-actin (A2066, 1:2000, Sigma-Aldrich, St. Louis, MO, USA). The bound antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and an ECL western blotting detection reagent (Bio-Rad, Hercules, CA, USA).
Anti pkc
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-PKC is a laboratory reagent used for the detection and quantification of Protein Kinase C (PKC) in biological samples. It is a highly specific antibody that binds to PKC, a family of enzymes involved in various cellular processes. The core function of Anti-PKC is to provide a reliable tool for researchers to study the expression, localization, and activity of PKC in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho pkc, by Santa Cruz Biotechnology (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Ecl western blotting detection reagent, by Bio-Rad (1 mentions)
Anti phospho erk sc 7383, by Santa Cruz Biotechnology (1 mentions)
Anti phospho pkc, by Cell Signaling Technology (1 mentions)
Anti phospho pdk1, by Cell Signaling Technology (1 mentions)
Anti pdk1, by Cell Signaling Technology (1 mentions)
Anti nf κb, by Cell Signaling Technology (1 mentions)
Anti phospho stat3, by Cell Signaling Technology (1 mentions)
Anti phospho nf κb, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by Abcam (1 mentions)
Hybond, by Cytiva (1 mentions)
Intercept blocking buffer, by LI COR (1 mentions)
Anti gsk3β, by Santa Cruz Biotechnology (1 mentions)
Nitrobind nitrocellulose membrane, by Santa Cruz Biotechnology (1 mentions)
Anti p27, by Santa Cruz Biotechnology (1 mentions)
Anti hunk, by Thermo Fisher Scientific (1 mentions)
Anti phospho mbp, by Merck Group (1 mentions)
11 protocols using anti pkc
1
Western Blot Analysis of Signaling Proteins
2
Antioxidant Protein Detection by Western Blot
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A total of 50 µg of total proteins was boiled in Laemmlie sample buffer for 5 min, separated by SDS‒PAGE and transferred to a NitroBind nitrocellulose membrane (Santa Cruz Biotechnology, Heidelberg, Germany). After blocking with Intercept Blocking Buffer (LI-COR Biosciences, Germany), antioxidant proteins are detected following membrane incubation with anti-G6PD, anti-NRF2 (Cell Signaling Technologies, Milan, Italy), anti-SOD1, anti-HO-1 (Santa Cruz Biotechnologies, Milan, Italy), anti-p38 and phosphorylated p38 (Santa Cruz Biotechnologies, Milan, Italy), anti-phosphorylated NRF2 (Ser40) (Thermo Fisher Scientific, Milan, Italy), anti-PKC (Santa Cruz Biotechnologies, Milan, Italy) or anti GSK-3β (Santa Cruz Biotechnologies, Milan, Italy). Actin was used as a loading control. After being washed three times with PBS containing 0.05% Tween-20 (PBS-T), the membranes are incubated with IRDye800/680-labelled goat anti-mouse/rabbit secondary antibodies (LI-COR Biosciences, Germany). Membranes are analysed by Odyssey Infrared Imager, and integrated intensities of fluorescence are used for densitometry analysis by using ImageJ software.
3
Western Blot Analysis of Signaling Proteins
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All cells were lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1% Triton X-100 with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific). Primary antibodies used for western blotting include: anti-phospho-MBP (EMD Millipore, 05-429), anti-MBP (LifeSpan BioSciences, LS-C312288/59980), anti-flag-M2 (Sigma-Aldrich, #F1804), anti-HUNK (Invitrogen PA5-28765), anti-p27 (Santa Cruz, sc-528), anti-phospho-PKC-substrates (Cell Signaling, #2261), anti-phospho-PKC (Santa Cruz, sc-271920), anti-PKC (Santa Cruz, sc-17769), and anti-β-tubulin (Santa Cruz, sc-55529). Imaging was performed on the Protein Simple FluorChem-R imaging system.
4
Seawater-induced Lung Injury Model
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Male Sprague–Dawley rats, weighing 180–220 g each, were obtained from the Animal Center of the Fourth Military Medical University. The study was approved by the Ethics Review board of the Fourth Military Medical University. All animal experiments were performed in accordance with the National Institute of Health’s guidelines for the care and use of laboratory animals (Publication No.85-23, revised 1985).
The formula seawater was prepared according to the composition of water from the East China Sea provided by Chinese Ocean Bureau: osmolality 1300 mmol/L, pH 8.2, specific gravity 1.05, NaCl 26.518 g/L, MgSO4 3.305 g/L, MgCl2 2.447 g/L, CaCl2 1.141 g/L, KCl 0.725 g/L, NaHCO3 0.202 g/L, and NaBr 0.083 g/L. The artificial seawater was confirmed to be sterile before endotracheal instillation. Anti-p-Ser368 of Cx43, anti-Cx43, anti-phospho-PKC, anti-PKC, and anti-β-actin antibodies for Western blot were purchased from Santa Cruz Biotechnology (Cell Signaling Inc). Staurosporine (STS, inhibitor of PKC) and phorbol myristate acetate (PMA, activator of PKC) were purchased from Beyotime (Nantong, China).
The formula seawater was prepared according to the composition of water from the East China Sea provided by Chinese Ocean Bureau: osmolality 1300 mmol/L, pH 8.2, specific gravity 1.05, NaCl 26.518 g/L, MgSO4 3.305 g/L, MgCl2 2.447 g/L, CaCl2 1.141 g/L, KCl 0.725 g/L, NaHCO3 0.202 g/L, and NaBr 0.083 g/L. The artificial seawater was confirmed to be sterile before endotracheal instillation. Anti-p-Ser368 of Cx43, anti-Cx43, anti-phospho-PKC, anti-PKC, and anti-β-actin antibodies for Western blot were purchased from Santa Cruz Biotechnology (Cell Signaling Inc). Staurosporine (STS, inhibitor of PKC) and phorbol myristate acetate (PMA, activator of PKC) were purchased from Beyotime (Nantong, China).
5
Western Blot Analysis of Abl Signaling
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Cells were lysed in 20 mM Tris pH 7.5, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 x complete protease inhibitor tablets (Roche Diagnostics, Germany), 1 mM sodium molybdate, 20 mM NaF, 10 mM sodium pyrophosphate, 20 mM β-glycerophosphate, 1 mM sodium vanadate. Equal protein amounts were separated by SDS PAGE and transferred onto nitrocellulose. Following antibodies were used: anti-c-Abl (AB3, Merck Biosciences, Germany), anti-pAblT735, anti-pCrkIIY221 (both New England Biolabs, Germany), anti-pAblY245, anti-β-actin (both Sigma Aldrich, Germany), anti-pAblY412, anti-GAPDH (both Abcam, UK), anti-CagA [44 (link)], anti-GST (Biomol Germany), anti-14-3-3 H8, anti-phospho-tyrosine (pY99), anti-TTK, and anti-PKC (all Santa Cruz Biotechnology, Germany). Membranes were imaged using the Molecular Imager ChemiDoc XRS system (BioRad, Germany). Where indicated, signals of protein bands were quantified using the ImageLab software (BioRad, Germany).
6
Quantifying Cx43, ERK, MEK, and PKC Phosphorylation
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Western blot was used to measure the phosphorylation of Cx43, ERK, MEK, and PKC. Proteins were extracted with radioimmunoprecipitation (RIPA) buffer (Millipore, Bedford, MA, USA) containing 0.1% protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor (Sigma-Aldrich), and centrifuged at 13,000 rpm for 20 min at 4 °C. The supernatants were collected, and the protein contents were determined using the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA), followed by 12% SDS-PAGE of 20 µg proteins and transfer to polyvinylidene difluoride membranes (Bio-Rad, Richmond, CA, USA). The membrane was incubated with anti-Cx43 (Sigma-Aldrich), anti-pERK1/2 (Cell signaling Technology, Beverly, MA, USA), anti-ERK1/2 (Cell signaling Technology), anti-pMEK (Cell signaling Technology), anti-MEK (Cell signaling Technology), anti-PKC (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-pPKC (Biovision Inc., Milpitas, CA, USA) antibodies overnight at 4 °C. Signals were detected with a chemiluminescence kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. The relative band intensity and phosphorylation status were quantified with an image-analysis program using Image J (NIH, Bethesda, MD, USA) or Scion Image (NIH).
7
Immunofluorescence Antibody Labeling Protocol
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The following primary antibodies were used in the course of this study: anti-Parvalbumine (EMD Millipore, Billerica, MA, MAB1572, 1:500), anti-eGFP (Genetex, GXT13970, 1:500), anti-PKC (Santa Cruz Biotechnology, Santa Cruz, CA, sc-209, 1:500), anti-humanNT3 (ThermoFisher Scientific, PA1-18385, 1:500), anti-alpha-tubulin (Genetex, Irvine, CA, GTX11304, 1:5000). The following secondary antibodies were used in the course of this study: anti-mouse-Alexa635 (ThermoFisher Scientific, A31574, 1:250), anti-rabbit-Alexa546 (ThermoFisher Scientific, A11081, 1:250), anti-chicken-Alexa488 (ThermoFisher Scientific, A11039, 1:250), anti-Rabbit IgG, HRP conjugated (Promega, W4011, 1:2000), anti-mouse IgG, HRP conjugated (Promega, Madison, WI, W4021, 1:2000).
8
Molecular Modulators of Cellular Signaling
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SB202190 (p38 inhibitor), U0126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor), LY294002 (AKT inhibitor) and R0318220 (PKC inhibitor) were purchased from Cell Signaling Technology (Beverly, USA). ML385 (Nrf2 inhibitor) and MG132 (NF-κB inhibitor) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Proanthocyanidin B1 (PB1), PB2 and PB4 were purchased from Sigma-Aldrich (St Louis, USA), with purity>98%. The primary antibodies including anti-Nrf2, anti-NQO1, anti-HO-1, anti-p-p38, anti-p38, anti-p-MEK, anti-MEK, anti-p-JNK, anti-JNK, anti-p-AKT, anti-AKT, anti-p-PKC, anti-PKC and anti-p65 were purchased from Santa Cruz Biotechnology. The antibodies including anti-IκB-α, anti-p-c-Jun, anti-c-Jun, anti-Lamin B, anti-α-tubulin, anti-β-actin, anti-PEPCK, anti-CPT1A, anti-GAPDH, and HRP-conjugated anti-mouse and anti-rabbit IgG secondary antibodies were purchased from Cell Signaling Technology (Beverly, USA). ImageJ software (NIH, Bethesda, USA) was used for quantitative analysis of each band. GAPDH, β-actin, Lamin B and α-tubulin were used as the loading control.
9
Affinity Purification of Protein Complexes
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The Ni2+-NTA agarose were obtained from QIAGEN (Dusseldorf, Germany) and Protein A+G Agarose beads were purchased from Beyotime Biotechnology (Shanghai, China). N-ethylmaleimide (NEM) were purchased from Sigma-Aldrich (St. Louis, MO). The complete protease inhibitor cocktail, the PhosSTOP phosphatase inhibitor cocktail and the In Situ Cell Death Detection Kit was purchased from Roche (Basel, Switzerland). Primary antibodies used in this research were listed below: anti-annexin A1 (sc-12740, 1:1000), anti-HA (sc-7392, 1:1000), anti-Flag (sc-166355, 1:1000), anti-α-tubulin (sc-100585, 1:2000), anti-β-actin (sc-47778, 1:2000), anti-PKC (sc-17769, 1:1000), anti-Bid (sc-11423, 1:1000) were purchased from Santa Cruz Biotechnology (Dallas, TX); anti-SUMO2/3 (#4971, 1:1000), anti-Myc (#2276, 1:1000), anti-Histone H3 (#4499, 1:2000), anti-cleaved caspase-3 (9664, 1:1000), anti-cleaved PARP (5625, 1:1000), anti-cleaved caspase-9 (#20750, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA); anti-SENP6 (HPA024376, 1:1000), anti-His (SAB1306085, 1:1000) were purchased from Sigma-Aldrich, and anti-TRPM7 (ab232455, 1:500) were purchased from Abcam (Cambridge, UK). All other general reagents were purchased from commercial suppliers and used as received.
10
Rat Myocardial Myoblast Culture
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Cell culture and reagents H9c2 cells, which are myoblasts cells from rat myocardium, were purchased from the American Type Culture Collection. H9c2 cells were cultured with Dulbecco's modi ed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (50 IU/mL)/streptomycin (50 μg/mL). A 0.25% (w/v) Trypsin-0.53 mM ethylenediaminetetraacetic acid (EDTA) solution was used to passage cells. Cells were cultured in humidi ed air with 5% CO 2 at 37°C. FBS and EDTA were purchased from Gibco (NY, USA). The 5,58,6,68-tetraethylbenzimidazolcarbocyanine iodide (JC-1), Ro-32-0432, and diphenyleneiodonium chloride (DPI) were purchased from Sigma (MO, USA). Dihydroethidium (DHE) was obtained from Thermo Scienti c (MA, USA). Anti-β-actin, anti-phospho-AMPK, anti-AMPK, anti-Nox-2, anti-Rac-1, anti-phospho-PKC, anti-PKC, anti-phospho-p53, anti-Bax, anti-Bcl2, anti-cytochrome c, anti-Na/K ATPase, and anti-PGC-1a were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Transduction Laboratories (CA, USA).
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