Cell lysates or immunoprecipitated products were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with primary antibodies overnight at 4°C. Blots were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Finally, blots were washed and visualized with ECL substrate (Pierce). Antibodies and dilutions were rabbit anti-HA (hemagglutinin)-tag monoclonal antibody (MAb) (C29F4) at 1:1,000 (Cell Signaling; 3724S), mouse anti-FLAG M2 antibody at 1:1,000 (Sigma-Aldrich; F1804), mouse anti-FUS MAb (4H11) at 1:200 (Santa Cruz; sc-47711), rabbit anti-CDK2 MAb (78B2) at 1:1,000 (Cell Signaling; S2546P), rabbit anti-DDX5 at 1:2,000 (Abcam; ab21696), and mouse antivimentin at 1:2,000 (Abcam; ab8978). Secondary antibodies were anti-mouse IgG (Santa Cruz; SC-2031) and anti-rabbit IgG (Pierce), both at 1:2,000.
Mouse anti flag m2 antibody
Manufactured by Merck Group
Sourced in United States, Japan
The Mouse anti-FLAG M2 antibody is a monoclonal antibody that specifically recognizes the FLAG peptide sequence (DYKDDDDK). It is a widely used tool in molecular biology and biochemistry research for the detection and purification of recombinant proteins tagged with the FLAG sequence.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Hiseq 4000, by Illumina (4 mentions)
Flag peptide, by Merck Group (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Goat anti mouse igg h l hrp conjugate, by Bio-Rad (2 mentions)
Image lab 4, by Bio-Rad (2 mentions)
Mouse anti actin antibody, by Cell Signaling Technology (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Mmachine t7 ultra kit, by Thermo Fisher Scientific (2 mentions)
Goat antimouse lgg h l alexa 647 antibody, by Thermo Fisher Scientific (2 mentions)
Accuri c6 cytometer, by BD (2 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
Anti β actin, by Abcam (2 mentions)
Aav tbg pi cre rbg, by Addgene (2 mentions)
Anti v5 antibody, by Thermo Fisher Scientific (2 mentions)
Pcag cre, by Addgene (2 mentions)
Mouse pm20d1 flag, by Addgene (2 mentions)
109 protocols using mouse anti flag m2 antibody
1
Western Blot Analysis of Protein Interactors
2
Western Blot Analysis of Protein Targets
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Samples were denatured in sample buffer and fractionated using SDS-PAGE gel. Then blots were transferred to polyvinylidene difluoride membranes (Immobilon-P, Merck Millipore), and reacted with mouse anti-FLAG M2 antibody (1:2000; Sigma, F1804), anti-V5 antibody (1:5000; abcam, ab27671), anti-tubulin antibody (1:2000; Sigma, T6199) or rabbit anti-histone H3 (1:2000; abcam, ab1791) for 1 h, subsequently with a horseradish peroxidase (HRP)-conjugated mouse IgG antibody (1:2000; Bio-Rad, immune-star anti-mouse HRP) or horseradish peroxidase (HRP)-conjugated rabbit IgG antibody (1:2000; Bio-Rad, immune-star anti-rabbit HRP) for 1 h. Clarity western ECL substrate (Bio-Rad) was used according to the manufacturer’s instructions to detect chemiluminescence. Fluorescent images were obtained using an ImageQuant LAS 4000 system (GE healthcare).
3
Selective Permeabilization of Cell Membranes
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HEK293 cells grown on glass coverslips were fixed with 3% paraformaldehyde in PBS for 10 min. and washed in PBS. For selective permeabilization of the plasma membrane, cells were incubated in buffer S (10 mM HEPES-KOH, pH 7.4; 0.3 M sucrose; 0.1 M KCl; 2.5 mM MgCl2; 1 mM EDTA, and 50 μg/ml digitonin (Sigma)) for 3 min on ice [24 (link)]. For permeabilization of all membranes, cells were incubated with 1% Triton X-100 in PBS for 10 min and blocked with 1% BSA in PBS. Mouse anti-FLAG M2 antibody (1:500, Sigma, #A8592) and rabbit anti-GFP antibody (1:200, Life Technologies, #A11122) were diluted in blocking solution and incubated for 1 hr. at room temperature. Primary antibody binding was visualized using fluorescent dye-conjugated secondary antibodies: goat anti mouse Alexa 568 (Life Technologies, #A11032) or goat anti rabbit Alexa 568 (Life Technologies, #A11036) incubated for 45 min. DAPI (Life Technologies) staining was used to visualize nuclei. The samples were mounted with ProLong Gold antifade reagent (Life Technologies) and visualized using inverted fluorescence microscopy (Leica DMI6000) or a Leica SP5 (II) laser scanning confocal microscope (Leica, Buffalo Grove, IL) equipped with 40X (1.30 NA) and 63X (1.4–0.6 NA) oil immersion lenses. Leica LAS AF Lite software was used for recording and image processing.
4
Characterization of Leukocyte Adhesion
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Mouse anti-FLAG M2 antibody (Sigma), HRP-conjugated anti-FLAG antibody (Sigma), APC PSGL-1 antibody (FLEG) (ThermoFisher), Human Fibronectin (ThermoFisher), DMSO (Sigma), Phalloidin Alex647 (ThermoFisher), RIPA buffer (Sigma), Phorbol 12-Myristate 13-Acetate (PMA, sigma), SLeX (Sigma), Biotinylated ECL (Vector Laboratories), PNGase F (New England Biolabs), Dihydrorhodamine 123 (DHR123) (ThermoFisher), ProLong Gold Antifade Mountant (ThermoFisher), HRP-conjugated streptavidin (ThermoFisher).
5
Antibodies and Reagents for Porcine Cell Study
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Rabbit polyclonal NP and NS1 antibodies were produced in our lab [35 (link)]. The commercial antibodies used are as follows. Goat anti-porcine IL-1β antibody (BAF681): R&D Systems (Minneapolis, MN, USA); rabbit anti-porcine caspase-1 (p20) antibody (PAB592Po01): Cloud-Clone Corp. (Houston, TX, USA); mouse anti-Myc-tag antibody (#2276), mouse anti-β-actin antibody (#3700), rabbit anti-DRP1 antibody (#8570) and rabbit anti-phospho-DRP1 (S616) antibody (#3455): Cell Signaling Technology (Beverly, MA, USA); mouse anti-FLAG M2 antibody (F3165): Sigma; IRDye 680RD donkey anti-rabbit (926-68073), IRDye 800CW donkey anti-mouse (926-32212) and IRDye 800CW donkey anti-goat (926-32214) antibodies: LI-COR Biosciences (Lincoln, NE, USA). The following reagents were used: lipopolysaccharide (LPS) (L3024) and N-acetyl L-cysteine (NAC) (A9165): Sigma; Necrostatin-1 (Nec-1) (BML-AP309): Enzo Life Sciences (Farmingdale, NY USA); Mdivi-1 (ab144589): Abcam (Cambridge, MA, USA).
6
In vitro Translation and Western Blot
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In vitro translation was performed using a PURESYSTEM classic II (BioComber, Tokyo, Japan). For the RNA template for in vitro translation, either a +1-peuA′-flag RNA (30 pmol)/fur-flag RNA (3 pmol) mixture or a +39-peuA′-flag RNA (30 pmol)/fur-flag RNA (3 pmol) mixture was used. The PURESYSTEM reaction mixture (20 µl) was incubated at 37°C for 2 h, and the reaction was then terminated by adding an equal volume of 2× SDS-PAGE sample buffer. The samples were separated on a 15% SDS-polyacrylamide gel, and the protein bands were transferred to a Clear Blot Membrane-P (Atto, Tokyo, Japan). The membrane was blocked with Tris-buffered saline with Tween 20 (TBST) containing 0.3% skim milk and incubated overnight at 4°C with mouse anti-FLAG M2 antibody (Sigma) diluted 1,000-fold with blocking solution. The membrane was then washed four times with TBST, incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse secondary antibody (GE Healthcare), diluted 20,000-fold with blocking solution, and washed four times with TBST. Immunoreactive bands were detected with an ECL Select Western Blotting Detection Reagent (GE Healthcare) and visualized with a LAS-3000 gel imager (Fujifilm, Tokyo, Japan)
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In vitro translation was performed using a PURESYSTEM classic II (BioComber, Tokyo, Japan). For the RNA template for in vitro translation, either a +1-peuA′-flag RNA (30 pmol)/fur-flag RNA (3 pmol) mixture or a +39-peuA′-flag RNA (30 pmol)/fur-flag RNA (3 pmol) mixture was used. The PURESYSTEM reaction mixture (20 µl) was incubated at 37°C for 2 h, and the reaction was then terminated by adding an equal volume of 2× SDS-PAGE sample buffer. The samples were separated on a 15% SDS-polyacrylamide gel, and the protein bands were transferred to a Clear Blot Membrane-P (Atto, Tokyo, Japan). The membrane was blocked with Tris-buffered saline with Tween 20 (TBST) containing 0.3% skim milk and incubated overnight at 4°C with mouse anti-FLAG M2 antibody (Sigma) diluted 1,000-fold with blocking solution. The membrane was then washed four times with TBST, incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse secondary antibody (GE Healthcare), diluted 20,000-fold with blocking solution, and washed four times with TBST. Immunoreactive bands were detected with an ECL Select Western Blotting Detection Reagent (GE Healthcare) and visualized with a LAS-3000 gel imager (Fujifilm, Tokyo, Japan)
7
Protein Expression and Detection
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Purified Fc- and DHD15-tagged E1E2 or E2-Fc proteins were separated by SDS-PAGE (6% or 8%) and stained with coomassie brilliant blue R-250 (Aladdin). For western blot detection, both supernatants and cell pellets were run on SDS-PAGE (8%) for separation and transferred onto a polyvinylidene difluoride (PVDF) membrance (Invitrogen). The membrane was probed with mouse anti-His tag antibody (1:1000 dilution; Proteintech) or mouse anti-Flag M2 antibody (1:1000 dilution; Sigma) followed by the HRP-conjugated rabbit anti-mouse IgG secondary antibody (Proteintech). After washing three times with the buffer (25mM Tris, 150mM NaCl, pH 7.4, 0.05% Tween-20), the membrane was incubated with Diaminobenzidine (DAB, Sigma) for detection.
8
Immunoprecipitation of ZO-2 Mutants
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Immunoprecipitations were done following a standard procedure previously described [10 (link)]. Transfected FLAG3-hZO-2 construct and the mutants FLAG3-hZO-2-K759R, -K992R, and -K730R were immunoprecipitated with 1 μg/μL of a mouse anti-FLAG M2 antibody (Cat. F1804, Sigma Aldrich, Saint Louis, MO, USA). For the experiments done to test the ubiquitination of ZO-2 mutants, 24 h after transfection, the cells were incubated for 2 h with 50 μM of PR616 (Cat. SI9619, Life Sensors, Malvern, PA, USA), a permeable inhibitor of ubiquitin/ubiquitin-like proteases that protects polyubiquitylated proteins from degradation [29 (link)].
9
Immunoblotting with Protein Lysates
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For immunoblotting, cells were lysed in 50 mM Tris buffer, pH 7.5 containing 0.1% TritonX (Fisher Scientific) and supplemented with protease inhibitor cocktail (Roche). Protein lysate concentrations were normalized by BCA assay (Thermo Fisher). Lysates were boiled for 5 min in Laemmli buffer with 100 mM DTT before loading onto SDS-PAGE gels. Proteins were transferred from gel slabs to nitrocellulose and blotted using mouse anti-FLAG M2 antibody (Sigma) or rabbit anti-PDIA4 antibody (Protein Tech) and visualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences).
10
Detecting Atg12 Phosphorylation via Phos-tag SDS-PAGE
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Endogenous SF-tagged Atg12 purified from cells expressing either wild-type Atg1 or catalytically inactive Atg1D211A was run on a 6% Phos-Tag SDS-PAGE gel containing 50 μM Phos-tag acrylamide (Alpha Laboratories) in 1x Tris glycine running buffer. The gel was washed twice in Protein Transfer Buffer containing 10 mM EDTA and once in EDTA free Transfer Buffer before western blotting using a PVDF membrane. SF-Atg12 was detected using a mouse anti-FLAG M2 antibody (Sigma) and goat anti-mouse IgG (H+L) HRP conjugate (Bio-Rad).
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