Jurkat cells were transfected with synthetic miR-27 or a scrambled control as described above, and harvested after 48 hours. Transfected cells were activated according to (Sawasdikosol, 2010 ), lysed directly in 1× Laemmli sample buffer, and separated on a 10% SDS-PAGE gel, then transferred to nitrocellulose membrane. Anti-CD3 antibody (OKT3) was from eBiosciences (#16-0037), and secondary rabbit anti-mouse IgG antibody was from Southern Biotech (#6170). Typically 100,000 cells were loaded in each lane. Primary antibodies used for Western blot (Cell Signaling) include anti-phospho-JNK (#4668), anti-total-JNK (#9258), anti-phospho-p38 (#4511), anti-total-p38 (#9212), anti-phospho-ERK (#4370), anti-total-ERK (#4695) and anti-phospho-tyrosine (#9416).
Anti total jnk
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-total JNK is a lab equipment product provided by Cell Signaling Technology. It is an antibody that recognizes the c-Jun N-terminal kinase (JNK) protein, which is a member of the mitogen-activated protein kinase (MAPK) family. The antibody can be used to detect and quantify the total levels of JNK in cell or tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho jnk, by Cell Signaling Technology (10 mentions)
Anti total p38, by Cell Signaling Technology (7 mentions)
Anti p jnk, by Cell Signaling Technology (5 mentions)
Anti total erk, by Cell Signaling Technology (4 mentions)
Anti phospho p38, by Cell Signaling Technology (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Anti phospho erk, by Cell Signaling Technology (3 mentions)
Anti p p38 mapk, by Cell Signaling Technology (3 mentions)
Anti phospho akt, by Cell Signaling Technology (3 mentions)
Anti total nfκb, by Cell Signaling Technology (3 mentions)
Anti total akt, by Cell Signaling Technology (3 mentions)
Anti total erk1 2, by Cell Signaling Technology (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Anti p smad2 3, by Cell Signaling Technology (2 mentions)
Sds page gel, by Bio-Rad (2 mentions)
Anti smad2 3, by Cell Signaling Technology (2 mentions)
Anti p erk, by Merck Group (2 mentions)
Anti col1a2, by Santa Cruz Biotechnology (2 mentions)
Anti total erk, by Merck Group (2 mentions)
19 protocols using anti total jnk
1
Regulation of T-cell Signaling Pathways
2
Western Blot Analysis of Cell Signaling Pathways
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Confluent cultures of C4, C9 and VMM12 cells in 6-well plates were placed in 1 ml fresh DMEM/10% FBS for times as indicated for specific experiments. Total cell lysates were prepared with RIPA buffer (Sigma Aldrich, St. Louis, MO) and were separated on 10% SDS-PAGE gels (Bio-Rad, Hercules, CA). Serum-free conditioned medium (from 48 h cultures) was TCA precipitated, resuspended in 30 µl of 2× Laemmli buffer, and separated on 10% SDS-PAGE gels. Proteins were transferred to a PVDF membrane and visualized with anti-Col1A2 (1:200, Santa Cruz, CA), anti-MMP-1 (1:5000, Millipore), anti-pERK (9101), anti-total ERK (9102), anti-pAKT (4058), anti-total AKT (9272), anti-pSmad 2/3 (9520), anti-Smad 2/3 (5678), anti-total JNK (9252), anti-p-p38 MAPK (9215), or anti-p38 MAPK (8690) (1:1000 for each, Cell Signaling, Beverly, MA), anti-p-JNK (1:2000, Cell Signaling), or anti-Smad 7 (1:1000, Thermo Scientific, Rockford, IL), followed by the appropriate secondary antibody, donkey anti-rabbit HRP (1:20,000) or donkey anti-mouse (1:10,000). Signal was detected with Western Lightning Plus ECL (PerkinElmer, Waltham, MA, USA).
3
Duoxa1 Overexpression in Mouse Cells
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Full-length wild-type Duoxa1 was amplified using PCR from mouse cDNA and ligated into the pMX-IRES-EGFP vector as pMX-Duoxa1 using the BamHI and XhoI (Enzynomics, Daejeon, Korea) sites. The following primers were used: Duoxa1-For, 5′-GCTAGGATCCATGGCTGCTCTTGGACACAC-3′ and Duoxa1-Rev, 5′-CGACTCGAGCAGGGAACAGTCGGACTCTTTG-3′. TRIzol reagent was obtained from Life Technologies (Carlsbad, CA, USA). A monoclonal β-actin (A5441) antibody and DAPI (D9542) were obtained from Sigma (St. Louis, MO, USA). Antibodies for anti-phospho-ERK-1/2, anti-total ERK-1/2, anti-phospho-p38, anti-total p38, anti-phospho-JNK, anti-total JNK, anti-phospho-IκB, anti-phospho-Akt, anti-Akt, anti-phospho-Src, anti-Src, and anti-Btk were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-Phospho-Btk (GTX61791) antibody was obtained from GeneTex (Irvine, CA, USA). Anti-c-Fos, anti-NFATc1, anti-IκB, and anti-PLCγ2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Duoxa1 antibody was obtained from Bioss Inc (BS-11433®, Wobun, MA, USA). Donkey anti-rabbit and anti-mouse immunoglobulin secondary antibodies were purchased from Enzo Life Sciences (Farmingdale, NY, USA).
4
JNK3 Overexpression and Signaling
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BHK and HuH-7 cells were transfected with 2, 4, and 6 μg
of pAAV-CAG-JNK3-GFP plasmids using lipofectamine 2000 (Thermo Fisher).
Cells were fixed at 24 h and pJNK and total JNK expression was studied
by immunoblotting and immunofluorescence using an anti-pJNK primary
mouse monoclonal antibody (1:1000, Cell Signaling Technology, Beverly,
MA) and anti-totalJNK (1:1000, Cell Signaling Technology, Beverly,
MA). A donkey anti-rabbit Alexa-546-conjugated antiserum (Invitrogen
ref A21202, 1:1000) was used for detection.
of pAAV-CAG-JNK3-GFP plasmids using lipofectamine 2000 (Thermo Fisher).
Cells were fixed at 24 h and pJNK and total JNK expression was studied
by immunoblotting and immunofluorescence using an anti-pJNK primary
mouse monoclonal antibody (1:1000, Cell Signaling Technology, Beverly,
MA) and anti-totalJNK (1:1000, Cell Signaling Technology, Beverly,
MA). A donkey anti-rabbit Alexa-546-conjugated antiserum (Invitrogen
ref A21202, 1:1000) was used for detection.
5
Arctigenin-Induced Cell Apoptosis Signaling
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Arctigenin (Fig. 1 ), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and 4´ ,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). LIVE/DEAD Viability/Cytotoxicity kit was purchased from ThermoFisher Scientific, Inc. (Waltham, MA, USA). PhiPhiLux-G1D2 Caspase-3/7 Assay Kit were purchased from OncoImmunin Inc. (Gaithersburg, MD, USA). Anti-cleaved caspase-3, -8, -9, anti-Fas, anti-PARP, anti-Bcl-2, anti-Bcl-xL, anti-Bax, anti-Bad, anti-Phospho-ERK, anti-total-ERK, anti-phospho-p38, anti-total-p38, anti-phospho-JNK, anti-total-JNK, anti-phospho-NFκB, anti-total-NFκB, anti-phospho-AKT, anti-total-AKT, anti-p53 and anti-β-actin antibodies were supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA).
6
Lipid Metabolism Regulation Assay
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Most chemicals used in this study were purchased from Sigma–Aldrich (St. Louis, MO, USA) including the followings: glucose, palmitate, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoilium bromide (MTT), DL-fluorocitric acid barium salt, Oil Red O and Eosin Y. Nile Red and Hematoxylin. BODIPY™-palmitate (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-caspase 3, anti-phospho-AKT, anti-total AKT, anti-phospho-GSK3b, anti-total GSK3b, anti-phospho-JNK, anti-total JNK, anti-phospho-P65, and anti-total P65 antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-actin and anti-tubulin antibodies were purchased from Bethyl Laboratory (Montgomery, TX, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. The catalog numbers of all reagents and antibodies were listed in Supplementary Table 2 of SI .
7
Quantification of MAPK Signaling in VSMCs
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VSMCs were lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/mL phenylmethyl-sulfonyl fluoride, and 2 µg/mL aprotinin). The suspension was incubated on ice and then centrifuged (14 000 g, 10 minutes, 4°C). Protein concentrations were measured by Bradford assay with bovine serum albumin as a standard. 60 µg protein supernatants were used for electrophoresis. Primary antibodies were used at the indicated dilutions as follows: LTCCα1C, 1 : 200 (Alomone); β-actin, 1 : 1000; anti-p-p38 MAPK, 1 : 1000; anti-total-p38 MAPK, 1 : 1000; anti-p-JNK, 1 : 1000; anti-total-JNK 1 : 1000; anti-p-ERK1/2 (1 : 1000); and anti-total-ERK1/2 (1 : 1000); all were from Cell Signalling Technology (Danvers, MA). The intensity of the bands was quantified by densitometry. Blots were representative of at least three experiments.
8
Autophagy Regulation in A. hydrophila Infection
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HKMs (1 × 107/mL) incubated with inhibitors or transfected with sc-siRNA or chop-siRNA were infected with A. hydrophila. The HKMs were lysed for CHOP at 2 h p.i., LC3B, Beclin-1, Atg 5 at 4 h p.i. and for total and phospho-JNK at 24 h p.i., resolved on SDS-PAGE and transferred to PVDF membrane. Membrane was blocked with 1% BSA in TBST for 1 h and subsequently incubated with anti-CHOP (1: 100, Cell Signaling Technology), anti-LC3B (1: 1000, Abcam), anti-Beclin-1 (1: 1000, Abcam), anti-Atg 5 (1: 1000, Abcam) and anti-total JNK and anti-phosphorylated JNK (Cell Signaling Technology, 1: 500 dilution), respectively, overnight at 4°C. Anti-GAPDH antibody (Santacruz, 1: 5000) was used for the normalization of Beclin-1, Atg 5, LC3B, and anti-β-actin antibody (Cell Signaling Technology, 1:10,000 dilution) was used for CHOP and JNK.
9
Protein Expression Analysis in Colon Tissues
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Colon tissues were lysed using a radioimmunoprecipitation assay (RIPA). The protein concentration was detected using the BCA protein assay kit (Life Technologies, Eugene, OR, USA). Thirty milligrams of the proteins were separated by electrophoresis on a 10% SDS polyacrylamide gel and then transferred to PVDF membranes. The samples were incubated with primary antibodies against phospho-ERK1/2 (Cell Signaling Technology, Boston, MA, USA), anti-total ERK1/2 (Cell Signaling Technology), phospho-JNK (Cell Signaling Technology), anti-total JNK (Cell Signaling Technology), phospho-p38 (Cell Signaling Technology), anti-total p38 (Cell Signaling Technology), phospho-NF-κB (Cell Signaling Technology), anti-total NF-κB (Cell Signaling Technology), and HSP90 (Cell Signaling Technology). Proteins were detected using the HRP-conjugated secondary antibody and the chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).
10
C2C12 Mesenchymal Cell Signaling Pathway
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The C2C12 mesenchymal cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The adenovirus-BMP2 (Adv-BMP2) and adenovirus-β-Gal (Adv-β-Gal) were provided by Dr. Jueren Lou (Institute of Biological Products, Shanghai, China). The Human TNF-α was purchased from PeproTech (300-01A; Rocky Hill, NJ). Real-time PCR was done via the ABI7900HT system using SYBR1Premix Ex TaqTM (DRR041A; Takara, Dalian, China). The anti-SATB2 (1:1000; SATBA4B10), anti-Lamin B (1:500; ab151735) and anti-TNF-α (1:600; ab34674) antibodies were obtained from Abcam (Cambridge, MA). The anti-GAPDH antibody (1:10000; sc-32233) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phospho-p44/42 ERK (1:1000; #4370) and anti-total-p44/42 ERK (1:1000; #4695), anti-phospho-p38 (1:1000; #4631) and anti-total-p38 (1:1000; #8690), and anti-phospho-JNK (1:1000; #4668) and anti-total-JNK (1:1000; #9252) antibodies, the anti-P65 (1:1000; #8442) and anti-beta-actin antibodies (1:10000; #3700) were purchased from Cell Signaling Technology (Danvers, MA).
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