The anti-TDP-43 antibody was described previously,66 (link) and the following additional primary antibodies were used: anti-iNOS (Abcam, Cambridge, MA, USA), anti-COX-2 (Epitomics, Burlingame, CA, USA), anti-GAPDH (Chemicon, Temecula, CA, USA), anti-phospho-MEK (S217/221) (Cell Signaling Technology, Beverly, MA, USA), anti-MEK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-ERK (Santa Cruz Biotechnology), anti-ERK (Santa Cruz Biotechnology), anti-phospho-JNK (Epitomics), anti-JNK (Santa Cruz Biotechnology), anti-phospho-p38 (Epitomics), anti-p38 (Santa Cruz Biotechnology), anti-phospho-c-Raf (S338) (Cell Signaling Technology), anti-c-JUN (Proteintech, Chicago, IL, USA), anti-phospho-Glycogen Synthase (S641) (p-GS) (Epitomics) and anti-MAP2 (Santa Cruz Biotechnology). The secondary antibodies used included horseradish peroxidase-conjugated sheep anti-mouse and anti-rabbit antibodies (Amersham Pharmacia Biotech, Peapack, NJ, USA). The antibody-bound proteins were visualized using an ECL detection kit (Amersham Biosciences, Piscataway, NJ, USA) or Alexa Fluor 488 (green) donkey anti-mouse IgG (Invitrogen, La Jolla, CA, USA).
Anti mek
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-MEK is a laboratory reagent used in various research applications. It functions as an inhibitor of the MEK (Mitogen-Activated Protein Kinase Kinase) enzyme, which is a key component of the MAPK signaling pathway. Anti-MEK is commonly utilized in cell-based assays and biochemical studies to investigate the role of the MAPK pathway in cellular processes.
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Lab products found in correlation
Anti erk, by Santa Cruz Biotechnology (7 mentions)
Anti phospho erk, by Santa Cruz Biotechnology (4 mentions)
Anti jnk, by Santa Cruz Biotechnology (3 mentions)
Anti p38, by Santa Cruz Biotechnology (3 mentions)
Anti phospho mek, by Santa Cruz Biotechnology (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti cox 2, by Abcam (1 mentions)
Anti map2, by Santa Cruz Biotechnology (1 mentions)
Ecl detection kit, by Cytiva (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti c jun, by Proteintech (1 mentions)
Anti inos, by Abcam (1 mentions)
Anti braf, by Santa Cruz Biotechnology (1 mentions)
Anti nras, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti craf, by BD (1 mentions)
Anti p erk, by Merck Group (1 mentions)
G box, by Syngene (1 mentions)
Anti p mek, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
9 protocols using anti mek
1
Immunoblotting Analysis of Protein Signaling
2
Western Blot Analysis of MAPK Pathway Proteins
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For SDS–PAGE analysis, the membranes were blocked with 5% low-fat milk in PBS Tween 20 (10%) for 30 min at room temperature. Membranes were then probed overnight at 4 °C with the appropriated primary antibodies: anti-ARAF (sc408, Santa Cruz, 1:500), anti-BRAF (sc5284, Santa Cruz, 1:2,000), anti-CRAF (#610151, BD Biosciences, 1:2,000), anti-NRAS (sc519, Santa Cruz, 1:2,000), anti-pMEK (#9121, Cell Signaling, 1:1,000), anti-MEK (sc219, Santa Cruz, 1:2,000), anti-pERK (M8159, Sigma, 1:2,000), anti-ERK (sc93, Santa Cruz, 1:2,000) and anti-βactin (A1978, Sigma, 1:5,000) antibodies. Signals were acquired using a CDD camera (G:BOX, Syngene). Uncropped western blotting pictures are shown in Supplementary Fig. 9 .
3
In Vivo and In Vitro Evaluation of Kinase Inhibitors
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RCC tumours were harvested on d62 from mice receiving daily oral drug treatment (sunitinib 40 mg/kg/d, axitinib 30 mg/kg/bid, pazopanib 100 mg/kg/bid, either as single agents or combined with PD-325901, 4 mg/kg/d). Without culturing in vitro, tumours were immediately lysed in cold RIPA buffer plus phosphatase inhibitors using sonication × 15 s on ice. Ren-01 cells (2 × 106) were plated in RPMI-1640 (10% FBS) in 6-well plates. Sixteen hr later cells were treated for 24 h with sunitinib (2 μM), axitinib (2 μM), or pazopanib (2 μM), either alone or in combination with PD-325901 (1 nM). Cells were washed × 2 with cold PBS and lysed in cold RIPA buffer containing phosphatase inhibitor cocktail (Sigma). Protein concentration was determined using BCA protein assay (Pierce Biotechnology). Twenty micrograms of protein were separated under reducing conditions on 4–15% gradient SDS-PAGE gels. Blots were probed with primary antibodies (anti-MEK, anti-phospho-MEK, anti-ERK, anti-phospho-ERK, from Santa Cruz Biotechnology) followed by enhanced chemiluminescence reagent (Amersham).
4
Comprehensive Western Blot Analysis
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Western blot analysis was performed as described previously [21 (link)]. The primary antibodies used for Western blot analyses included anti-phospho-Src (Tyr418) (Invitrogen, Carlsbad, CA, USA), anti-phospho-FAK (Tyr576) (Invitrogen), anti-FAK (Invitrogen), anti-GAPDH (Invitrogen), anti-phospho-EGFR (Tyr1068) (Cell Signaling, Beverly, MA, USA), anti-phospho-STAT3 (Tyr705) (Cell Signaling), anti-phospho-PI3K (Tyr458) (Cell Signaling), anti-AKT (Cell Signaling), anti-phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling), anti-SAPK/JNK (Cell Signaling), anti-phospho-Paxillin (Tyr118) (Cell Signaling), anti phosphor-p130Cas (Tyr410) (Cell Signaling), anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT3 (Santa Cruz Biotechnology), anti-PI3K (Santa Cruz Biotechnology), anti-phospho-MEK1/2 (Ser218/Ser222) (Santa Cruz Biotechnology), anti-MEK (Santa Cruz Biotechnology), anti-phospho-ERK (Tyr204) (Santa Cruz Biotechnology), anti-ERK2 (Santa Cruz Biotechnology), anti-Paxillin (Santa Cruz Biotechnology), anti-p130 Cas (Santa Cruz Biotechnology), anti-phospho-AKT (Ser473) (Millipore, Billerica, MA, USA)
5
miRNA Expression and Cellular Pathways
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The Affymetrix miRNA V3.0 array profiling platform was supplied by Affymetrix (Santa Clara, CA). The RNeasy Midi Kit was acquired from Qiagen (Valencia, CA). SYBR Premix EX TaqII, which was used for real time polymerase chain reaction (PCR), was purchased from Takara Korea Biomedical Inc. (Seoul, Korea). For the apoptosis assay, a FITC Annexin V Apoptosis Detection Kit was purchased from BD Biosciences Pharmingen (San Diego, CA). For the flow cytometric autophagy assay, Cyto-ID Green dye was acquired from ENZO Life Sciences, Inc. (Farmingdale, NY). Rabbit polyclonal anti-MEK, anti-ERK, anti-p21Cip1, and anti-p27Kip1 antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA), whereas anti–phospho-MEK (anti–p-MEK Ser217/221) and anti–phospho-ERK (anti–p-ERK, Thr202/Tyr204) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Life Technologies (Carlsbad, CA). The reagents for sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis were acquired from BioRad (Hercules, CA), while PLX4720 was obtained from Selleck Chemicals (Houston, TX).
6
Whole-Cell Protein Extraction and Western Blot Analysis
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To prepare whole-cell extracts, cells were scraped from the dishes and suspended in protein extraction buffer (100 mM Tris-Cl (Sigma-Aldrich) pH 7.8, 10 mM NaCl (Sigma-Aldrich), 10% glycerol (Sigma-Aldrich), 1 mM sodium orthovanadate (Sigma-Aldrich), 50 mM sodium fluoride (Sigma-Aldrich), and 1 mM phenymethylsulfonyl fluoride (Sigma-Aldrich). Equal amounts of protein were separated by electrophoresis on 10% SDS-polyacrylamide gels, followed by electrophoretic transfer to nitrocellulose membranes (EMD Millipore). Membranes were blocked in 5% nonfat dry milk (Amresco LLC) and 0.1% Tween-20 (Amresco LLC) in Tris-buffered saline and probed with the following primary antibodies; the anti-ERK, anti-JNK, anti−p38, anti-phospho-JNK, anti-MEK, anti-FLAG, anti-p53, anti-p21, anti-GSK3β, and anti-ACTIN (Santa Cruz Biotechnology), anti-phospho-ERK, anti-phospho-p38, anti-phospho-GSK3β (Ser9), and anti-phospho-MEK (Cell Signaling). Antibody-antigen complexes were incubated with anti-rabbit or anti-mouse-IgG-peroxidase conjugates (Santa Cruz Biotechnology), followed by detection using an enhanced chemiluminescence (ECL) kit (GE Healthcare Life Sciences).
7
Protein Expression Analysis of Signaling Pathways
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Tissues and cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid, 1% Triton-X100) containing a protease inhibitor cocktail (Sigma-Aldrich Co.). Cell extract protein amounts were quantified using the bicinchoninic acid protein assay kit (Beyotime). Equivalent amounts of protein (30 μg) were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene di fluoride membrane (EMD Millipore). Immunoblotting was performed using anti-ERK, anti-phospho-ERK, anti-MEK, anti-phospho-MEK, anti-MMP2, anti-MMP9, anti-E-cadherin, anti- N-cadherin, and anti-β-actin (Santa Cruz Biotechnology Inc.). Each specific antibody binding was detected with horseradish peroxidase-conjugated respective secondary antibodies (Amersham Biosciences, Amersham, UK) and enhanced chemiluminescence solutions (Amersham Biosciences).
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
Western Blot Protein Detection
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Cellular proteins were probed using the following antibodies: anti-ALDH1, anti-DR4, anti-DR5, anti-ERK, anti-phospho-ERK, anti-MEK, anti-phospho-MEK, and anti-β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The reaction was followed by probing with peroxidase-coupled secondary antibodies at dilutions ranging from 1:500 to 1:1000 (Beyotime Biotechnology), and binding results were visualized via enhanced chemiluminescence (Beyotime Biotechnology).
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