eEV and eEV-IL-3 were lysed in lysis buffer (RIPA buffer with proteinase inhibitors). Starting from the same EV particle number, protein samples were quantified by the Bradford method before performing Western blot (50 µg proteins/each sample were loaded). Anti-CD63, anti-CD81, antiHSP90, anti-GM130, anti-Bcl-2 (Abcam, Milan, Italy), anti-MEK1/2, (Cell Signaling, Danvers, MA, USA), anti-CD29, anti-caveolin-1, anti-p-eNOS-ser1177 (Invitrogen, Carlsbad, CA, USA), and anti-vinculin (Millipore, Milan, Italy) antibodies were used as primary antibodies. Appropriate HRP-conjugated secondary antibodies (BioRad, Milan, Italy) were used, and proteins were detected with Clarity Western ECL substrate (BioRad, Milan, Italy). Image Lab Software (BioRad Milan, Italy) instrument was used for densitometric analysis. Data are expressed as arbitrary unit. Ponceaus-staining has been used as input (EV protein content normalization).
Anti mek1 2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-MEK1/2 is a laboratory reagent that can be used to detect the presence and measure the levels of MEK1 and MEK2 proteins. MEK1 and MEK2 are part of the MAPK signaling pathway, which plays a role in various cellular processes. The anti-MEK1/2 product provides researchers with a tool to study the expression and activity of these proteins in experimental systems.
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Lab products found in correlation
Anti phospho mek1 2, by Cell Signaling Technology (22 mentions)
Anti erk1 2, by Cell Signaling Technology (21 mentions)
Anti p mek1 2, by Cell Signaling Technology (13 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (10 mentions)
Anti p erk1 2, by Cell Signaling Technology (9 mentions)
Anti akt, by Cell Signaling Technology (9 mentions)
Anti phospho akt, by Cell Signaling Technology (6 mentions)
Anti gapdh, by Cell Signaling Technology (5 mentions)
Anti phospho mek1 2 ser217 221, by Cell Signaling Technology (4 mentions)
Anti ras, by Cell Signaling Technology (4 mentions)
Anti stat3, by Cell Signaling Technology (4 mentions)
Anti p70s6k, by Cell Signaling Technology (3 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Anti p38, by Cell Signaling Technology (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti e cadherin, by Cell Signaling Technology (3 mentions)
Anti caspase 3, by Cell Signaling Technology (3 mentions)
Anti vimentin, by Cell Signaling Technology (3 mentions)
Anti erk, by Cell Signaling Technology (3 mentions)
56 protocols using anti mek1 2
1
Extracellular Vesicle Protein Profiling
2
Western Blot Analysis of JAK2 Signaling
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Leukemic cells were lysed in lysis buffer supplemented with freshly added protease and phosphatase inhibitors. 25μg (BCA method; Thermo Scientific) lysate was loaded on 10% mini protean precast gels (BioRad, Veenendaal, Netherlands), and transferred to a nitrocellulose membrane (Biorad). Primary antibody incubation was performed according to manufacturer's protocol. Anti-JAK2 (#3230), anti-phospho-JAK2-Tyr1007 (#4406), anti-phospho-STAT5-Tyr694 (#9351), anti-phospho-STAT1-Tyr701 (#9167), anti-Stat1 (#9175), anti-phospho-MEK1/2-Ser217/221 (#9154), anti-MEK1/2 (#4694), anti-phospho-Erk1/2-T202/204 (#4370), anti-Erk1/2 (#91078), and anti-αTubulin (#2144) were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA). Anti-β-actin (ab6276) was obtained from Abcam (Cambridge, UK), and anti-STAT5 (sc-835) from Santa Cruz (Heidelberg, Germany). Blots were stained with secondary antibodies (IRDye 680RD- or 800CW-labelled anti-rabbit and IRDye 680RD- or 800CW-labelled anti-mouse; Li-Cor Biosciences, Leusden, Netherlands) and scanned using the Odyssey infrared imaging system (Li-Cor Biosciences). To reprobe membranes, they were stripped in NewBlot Nitrocellulose stripping buffer (Li-Cor Biosciences) according to manufacturer's protocol. BCR-JAK2, PAX5-JAK2 and TERF2-JAK2 proteins were separated from wildtype JAK2 based on size (~94, 57, 95 and 125 kDa, respectively).
3
CTCL Cell Signaling Protein Analysis
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1 × 106 CTCL cells were lysed for 10 min in ice-cold RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, 0.5% Na-Desoxycholat, 0.1% SDS, 2 mM EDTA, 25 mM NaF, 0.2 mM NaVO4, 1 mM DTT, and complete protease inhibitor cocktail from Roche). Lysates were separated by SDS-PAGE and proteins were blotted onto a nitrocellulose membrane (Amersham Biosciences) followed by blocking with 5% BSA in PBS/Tween (0.05% Tween-20 in PBS). The following antibodies were used: anti-phospho-ERK (P-p44/p42 (Tyr202/204); Cell Signalling), anti-ERK2 (C-14; Santa Cruz), anti-phospho-MEK1/2 ((Ser217/212); Cell Signalling), anti-MEK1/2 (Cell Signalling), anti-Mcl-1 (S-19; Santa Cruz), anti-tubulin (Sigma-Aldrich).
4
Signaling Pathway Protein Analysis Protocol
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The following reagents and antibodies were obtained commercially: MG-132 (Calbiochem), anti-Akt1, anti-phospho-Akt1 (Thr308, Ser473), anti-p44/42 (Erk1/2), anti-phospho-p44/42 (Erk1/2) (Thr202/Tyr204), anti-MEK1/2, anti-phospho-MEK1/2 (Ser 217/221), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-p70S6K, anti-phospho-p70S6K (Thr389), anti-Rictor, anti-Raptor, anti-PRAS40, anti-phospho-PRAS40 (Thr246), anti-4E-BP1, anti-phospho-4E-BP1 (Thr 70), anti-FoxO1, anti-phospho-FoxO1 (Thr24 and Ser256), anti-FoxO3a, anti-phospho-FoxO3a (Ser253 and Thr32), anti-TSC2, anti-phospho-TSC2 (Thr1462), anti-SGK1, anti-SGK3 (Cell Signaling Technology), anti-p21Cip1 (Santa Cruz Biotechnology); anti-p27Kip1 (BD Biosciences), anti-cyclin D1 (MBL), and anti-β-tubulin (Sigma). anti-phospho-FoxO3a Ser314 antibodies were a generous gift from Dr. Michael E. Greenberg (Harvard Medical School, Boston).
5
Evaluating Cellular Stress Markers in Breast Cancer Cell Lines
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MDA-MB-468 cells and MCF-7 were treated with the indicated concentrations of ChPL for 24 h, and Western blot analysis was performed according to the previously published procedure (Kawiak et al., 2012b (link)). The preparation of cytosolic and mitochondrial fractions for cytochrome c release evaluation was performed as previously described (Kawiak et al., 2012b (link)). The following specific primary antibodies were used: anti-β-actin (1:1,000) (Cell Signaling, Danvers, MA, USA), anti-Bcl-2, anti-Bak, anti-Bax, and anti-Mcl-1 (1:250) (Santa Cruz, Heidelberg, Germany), anti-ERK1/2, anti-MEK1/2, anti-p-ERK1/2, and anti-p-MEK1/2 (1:1,000) (Cell Signaling), anti-cytochrome c (1:5,000) (Abcam, UK), and anti-HSP60 (1:1,000) (Cell Signaling). Membranes were incubated with primary antibodies overnight at 4°C after which a 1-h incubation with HRP-conjugated secondary antibodies (1:2000) (Cell Signaling) was carried out. Protein levels were determined by chemiluminescence (ChemiDoc; Bio-Rad, Waltham, MA, USA) with a HRP substrate (Thermo Scientific, MA, USA).
6
Western Blotting Analysis of Signaling Pathways
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Whole‐cell lysates were prepared in M‐PER buffer supplemented with protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), resolved by SDS/PAGE, and blotted with indicated antibodies. SuperSignal West Pico Chemiluminescent Substrate and SuperSignal Western Blot Enhancer (Thermo Fisher Scientific) were used to enhance blotting signal when needed. The following antibodies were used in this study: anti‐MAP4K4, anti‐phospho‐ERK1/2, anti‐phospho‐JNK, anti‐JNK, anti‐phospho‐p38, anti‐p38, anti‐AKT, anti‐phospho‐AKT, anti‐MEK1/2, anti‐phospho‐MEK1/2, anti‐RAS and anti‐PP2A‐C (Cell Signaling Technology, Danvers, MA, USA); anti‐ERK1/2, anti‐GAPDH, anti‐β‐actin, anti‐PP2A‐B56β, and anti‐MKP3 (Santa Cruz Biotechnology, Dallas, TX, USA); anti‐6Χ HIS (Immunology Consultants Laboratory, Inc., Lake Oswego, OR, USA). Erlotinib and trametinib were purchased from Selleck Chemicals (Houston, TX, USA). The protein phosphatase 2 (PP2A) inhibitor okadaic acid (OA) was purchased from Sigma Aldrich. The PP2A activator FTY720 was purchased from Cayman Chemical (Ann Arbor, MI, USA). MAP4K4 inhibitor PF‐06260933 was purchased from Tocris Bioscience (Avonmouth, Bristol, UK). EGF was obtained from Thermo Fisher Scientific.
7
Immunoprecipitation and Nuclease Assays
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Immunoprecipitation of HA-tagged proteins was performed as described [20 (link)]. Immunoprecipitation of endogenous proteins was performed as described [18 (link)] using 100 μg of crude nuclear or 175 μg refined cytoplasmic or nuclear extracts. S1 nuclease protection assay was performed as described [20 (link)]. Monoclonal and polyclonal HA antibodies (Cat#s MMS-101R and PRB-101C, respectively, Covance) were used for both IP (3 μL) and WB (1:1000). Anti-CPSF73, anti-Symplekin, and anti-CPSF100 antibodies (1:1000) were described previously [18 (link), 26 (link)]. Commercial anti-Dcr-2 (Abcam ab4732), anti-Actin (Abcam ab8227), anti-H3 (Cell Signaling 4499), and anti-MEK1/2 (Cell Signaling 8727) were used at manufacturer recommended concentrations. The anti-R2D2 antibody was a generous gift from the Siomi lab [27 (link)].
8
Molecular Mechanisms of Zoledronate-Induced Cell Death
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Anti-NF-κB, anti- Bcl-2, anti-phospho-Raf-1, anti-Akt, anti-vimentin, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti- cleaved PARP, anti-caspase-3, anti-Ras, anti-MEK1/2, anti-p-MEK1/2, anti-ERK1/2, anti-p-ERK1/2, anti-p-Akt(Ser473), and anti-E-cadherin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and anti-γ-H2A histone family member X antibodies were obtained from Millipore (Billerica, MA, USA). ZOL was purchased from Sigma–Aldrich (St. Louis, MO, USA). For in vitro experiments, ZOL was dissolved in PBS to prepare a 2 mM stock solution and stored at −20 °C.
9
EGFR and Downstream Pathway Activation Analysis
10
Cell Fractionation and Immunoblotting
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Subcellular fractions from cultured HEK293T cells were obtained using a cell fractionation kit (Cell Signaling Technology, 9038) according to the manufacturer's protocol. Cytoplasm, nuclear, and whole-cell extracts were separated by electrophoresis through 10%–20% Tris-glycine, and the resulting membrane was immunoblotted with anti-MEK1/2 (Cell Signaling Technology, 8727), anti-histone H3 (Cell Signaling Technology, 9715), anti-α-tubulin (Sigma, T9026), anti-β-tubulin (Abcam, ab15568), and anti-SET8 (Active Motif, 61009).
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