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14 protocols using p c jun

1

Western Blotting of Cell Signaling Proteins

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Western blotting was performed using whole cell lysates. Aliquots of total protein (20-50 μg per lane) were electrophoresed on a 10% SDS-polyacrylamide gradient gel and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were incubated at 4 °C overnight with anti-GAPDH, MYC, c-Jun, p-c-Jun, c-Fos, p-c-Fos, JNK, p-JNK, or Igκ monoclonal antibody (all purchased from Abcam, Cambridge, MA). After rinsing in buffer wash, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX) diluted 1:10,000-30,000, followed by development with enhanced chemiluminescence reagents (Amersham, Little Chalfont, UK).
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2

Immunohistochemical Analysis of Cellular Markers

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The expression levels of Ki-67, cleaved caspase-3, Beclin1, LC3B-II, CHOP, GRP78, p-JNK, and p-c-jun in tumor tissues were measured by IHC analysis according to the protocols previously described [25 (link)]. Briefly, 4-mm consecutive sections were deparaffinized in xylene, rehydrated in a graded ethanol series, and submerged in EDTA antigenic retrieval buffer for 15 min in a microwave oven. The sections were treated with 3% hydrogen peroxide in absolute methanol for 20 min to block endogenous peroxidase activity. Then, 5% BSA was applied for 15 min to prevent non-specific binding. The sections were incubated overnight at 4 °C with primary antibodies. Ki-67 (1:150), cleaved caspase-3 (5 μg/ml), Beclin1 (1:200), LC3B-II (1 μg/ml), CHOP (1:100), GRP78 (1 μg/ml), p-JNK (1:100) and p-c-jun (1:100) were purchased from Abcam (Cambridge UK). After incubation with the secondary antibody, the visualization signal was developed with 3,30-diaminobenzidine tetrachloride.
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3

Quantitative Western Blot Analysis

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After 72 h, the HSFs were washed with PBS and lysed in lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) supplemented with proteinase and phosphatase inhibitor cocktails on ice. The lysates were collected after centrifugation at 12,000g for 30 minutes at 4°C. The BCA protein quantification kit (ThermoFisher, USA) was used to quantify the total protein of the extracted cells. 20 μg sample protein from each group was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane and blocked with 5% skimmed milk powder blocking solution at room temperature for 1 h. Then, the membranes were incubated at 4°C overnight with the antibodies for α-SMA (Immunoway, USA), Smad3 (Abcam, USA), p-Smad3 (Abcam, USA), c-FOS (Abcam, USA), p-c-FOS (Abcam, USA),c-Jun (Abcam, USA), p-c-Jun (Abcam, USA), and GAPDH (Abcam, USA). After washed with TBS, the membranes were added secondary antibody (1 : 10 000) and shook gently at room temperature for 40 minutes, and ECL was added to the membrane to react for 3–5 minutes, and the film was exposed. The density of the bands was normalized to β-actin and quantified with Image software. Immunofluorescence was assessed using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).
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4

Quantifying Protein Expression in Cells

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Total protein was isolated from cultured cells using a lysis buffer supplemented with protease and phosphatase inhibitors. The protein concentration was measured using a protein assay kit (Bio-Rad, Hercules, CA, USA). An equivalent of 30 μg protein extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF). The membranes were incubated with the following primary antibodies: NF-κB p65, Pp65, p-c-Jun, c-Jun, β-Actin, pAMPK, AMPK, ACSS2, UCP2, PPARγ, pIκBα and IκBα (Abcam, Shanghai, China). After incubation with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Beyotime, Shanghai, China) at a dilution of 1:10,000 for 1 h, the proteins were finally visualized using a Luminata Forte Enhanced Chemiluminescence Kit (Millipore, Billerica, MA, USA) and detected by Imager 600 (Amersham, Switzerland). The band intensities were analyzed using ImageJ 1.54b (NIH).
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5

Primary Astrocyte Culture Protocols and Reagents

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The 2-Chloroethanol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Ningbo, China). Reagents for the primary culture of astrocytes were purchased from Biological Industries (Beit-Haemek, Israel). The quantitative real-time (RT)-PCR assay kit was purchased from Takara, Japan. The enhanced chemiluminescence (ECL) plus kit, bicinchoninic acid (BCA) protein assay kit and NE-PER™ nuclear and cytoplasmic extraction reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). SB202190, pyrrolidine dithiocarbamate (PDTC), and SR11302 were purchased from Selleck (Houston, TX, USA) and APExBIO (Houston, TX, USA). Primary antibodies against MMP-9, p65, IκBα, c-Jun, c-Fos, p-c-Jun, p-c-Fos, p-IκBα, and LaminB were products of Abcam (Cambridge, UK) and Cell Signaling Technology (Beverly, MA, USA). Antibodies against glial fibrillary acid protein (GFAP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β-actin were obtained from Millipore (Billerica, MA, USA), Proteintech (Wuhan, China), and ABclonal (Wuhan, China), respectively. The secondary antibodies conjugated with Alexa Fluor 488 or tetramethylrhodamine (TRITC), RIPA Lysis Buffer, and DAPI were obtained from Beyotime Biotechnology (Shanghai, China).
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6

Molecular Characterization of HNSCC Cells

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Patient-derived HNSCC tumor cells were collected and processed as previously described [9 (link), 47 (link), 48 (link)]. Retroviral vector pMSCV-Blasticidine is a gift from Dr. Mathijs Voorhoeve (Duke-NUS Medical School, Singapore). GNA13 is cloned in pMSCV-Blast vector as described earlier [27 (link)]. Stable cell lines expressing either pMSCV-Vector or GNA13 are created as described earlier [27 (link)]. ShRNAs targeting GNA13 were cloned in modified pRetro-Super vector (gift from Dr. Mathijs Voorhoeve) and stable cell lines were generated using blasticidine selection as described [27 (link)]. RPMI or DMEM complete media with 10% FBS and 1% penicillin/streptomycin (GIBCO) is used to maintain the cell lines. For Western blots, antibodies used were as follows: Gα13 (ST1629, San Diego, CA, USA), α-tubulin (010M4813, Sigma, St Louis, MO, USA), p-cJun (Thr91, ab28853, Abcam, Cambridge, UK) total cJun (9165), p-ERK1/2 (Thr202/Tyr204, 9101), Total ERK1/2 (9102), and total IκB-alpha (4812) (Cell Signaling, Boston, MA, USA) and p-IκB-alpha (Ser32, Ma5-15087, Thermoscientific, Rockford, IL, USA). Gα13 antibody used for immunohistochemistry was purchased from Sigma (HPA010087).
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7

Apoptosis and Autophagy Pathway Analysis

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XAG (HLPC ≥98%, MW: 392.49) was synthesized as previously described [22 (link)]. Specific antibodies against cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, cleaved caspase-12, cleaved PARP, Bcl-2, Bak, Bax, LC3B-II, p62/SQSTM1, Beclin-1, Atg5, p-JNK, JNK, p-c-jun, c-jun, Ki-67, CHOP, GRP78, ATF6, p-eIF2α, IRE1α were purchased from Abcam (Cambridge, UK), specific antibody against cytochrome C was obtained from Cell Signaling (Danvers, MA, USA). Antibody against COX-IV was purchased from Abcam (Cambridge, UK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and goat anti-rabbit immunoglobulin horse radish peroxide (IgG-HRP) or anti-mouse IgG-HRP were obtained from Beyotime Biotechnology Co. Ltd. (Shanghai, China). Fluorescent antibody against LC3 was purchased from Boster Biological Technology Co. Ltd. (Wuhan, China).
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8

Signaling Pathway Antibody Analysis

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The following antibodies were used: Actin, p-Met, c-MET, BCL-XL, FADD, p-Bad, bim, cleaved caspase-8, cleaved caspase-3, Phospho-p44/42 MAPK (Erk1/2), p44/42 MAPK (Erk1/2), p-c-jun, p-AKT, AKT, p-Raf, c-Raf Phospho-MEK1/2, MEK1/2 (Cell Signaling, Frankfurt, Germany); caspase-8, JNK1, and p-JNK (Santa Cruz, Heidelberg, Germany); CK19, c-jun, and p-c-jun (Abcam, Cambridge, UK); c-MET neutralizing antibody (Sigma-Aldrich, Munich, Germany); and the secondary antibodies for goat anti-rabbit and goat anti-mouse (LI-COR Biosciences GmbH, Bad Homburg, Germany).
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9

Immunohistochemical Analysis of Liver Tissues

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Sections (2 μm) of livers (fixed in 4% paraformaldehyde and paraffin-embedded) were stained with Hematoxylin/Eosin or various antibodies. Incubation in Ventana buffer and staining was performed on a NEXES immunohistochemistry robot (Ventana Instruments) using an IVIEW DAB Detection Kit (Ventana) or on a Bond MAX (Leica). Immunostainings were performed as described before (Wolf et al., 2014 (link)) with antibodies against the following proteins: Ki67, 1:200 dilution (SP6, NeoMarkers / Lab Vision Corporation); γH2AX, 1:300 dilution (Novus Biologicals); p-cJUN, 1:100 (Abcam); cleaved-Caspase8, 1:500; p-CHK1, 1:50 and p-CHK2, 1:500 (Novus Biologicals). For virtual microscopy and archiving, histological and immunohistochemical images were digitalized using a Nano Zoomer C9600 Virtual Slide Light microscope scanner by Hamamatsu using NDP, View Software, version 1.2.36. Alternatively, for quantification of stainings, slides were scanned using a SCN 400 slide scanner (Leica) and analyzed using Tissue IA image analysis software (Slidepath, Leica).
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10

Testicular Protein Expression Analysis

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Proteins were extracted from the testicular tissue using a lysis buffer. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were transferred onto a nitrocellulose film. After blocking, the membrane was treated with primary anti–HO–1 antibody (1:500; Cell Signalling, Danvers, MA, USA), p-c-Jun (1:1000; Abcam, Cambridge, MA, USA), c-Jun (Abcam), tubulin (1:1000; Abcam), and-actin (1:1000; Abcam). The membrane was thoroughly cleansed, incubated with an HRP-conjugated antibody, and detected using an ECL kit (Pierce Biotechnology, Rockford, USA).
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