Protein lysates were prepared in the same manner as described above. Between 50 and 100 μg total protein were separated on NuPAGE Novex 4–12% bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad, 162-0115, Hercules, CA, USA) for probing using the iBlot system (Invitrogen). Membranes were incubated in TBS blocking buffer (25 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) with 5% w/v skim milk powder for 2 h at room temperature before incubation with primary antibodies (anti-FLAG (Clone M2, Sigma); anti-HA (Sigma); anti-Myc (Sigma); anti-VSVG (Clone 16B12, Sigma); anti-GroEL2, and anti-Ag85) diluted in TBS with 1% BSA overnight. All antibodies were used at a dilution of 1:1,000 except anti-GroEL2 (1:50,00) and anti-Ag85 (1:3,000). Membranes were washed five times in TBS (15 min each at room temperature) before incubating with appropriate secondary antibody for 1 h at room temperature. Membranes were developed using the colorimetric horseradish peroxidase substrate 4-chloro-1-naphthol (4CN) (Opit-4CN Kit by Bio-rad). Antibodies against GroEL2 and Ag85 were generous gifts from Dr J.M. Chen as part of the National Institutes of Health, National Institute of Allergy and Infectious Diseases, contract HHSN266200400091c entitled ‘Tuberculosis Vaccine Testing and Research Materials,' awarded to Colorado State University).
Anti flag clone m2
Manufactured by Merck Group
Sourced in United States, Canada
The Anti-FLAG (clone M2) is a laboratory reagent used in various research and analytical applications. It is a monoclonal antibody that specifically binds to the FLAG peptide tag, which is a commonly used protein tag for recombinant protein expression and purification. The antibody can be used to detect, localize, and purify FLAG-tagged proteins.
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Lab products found in correlation
Anti myc clone 9e10, by Merck Group (4 mentions)
Anti ha clone ha 7, by Merck Group (4 mentions)
Anti α tubulin, by Santa Cruz Biotechnology (3 mentions)
Hrp conjugated anti mouse igg, by Cell Signaling Technology (3 mentions)
Ecl prime, by GE Healthcare (3 mentions)
Anti strepii, by Merck Group (3 mentions)
Anti 5his, by Merck Group (3 mentions)
Mouse secondary antibodies, by Merck Group (3 mentions)
Protran, by Cytiva (3 mentions)
Anti ha, by Merck Group (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Anti lmp1 clone s12, by Cell Signaling Technology (2 mentions)
Anti survivin clone 71g4b7, by Cell Signaling Technology (2 mentions)
Anti epha2 clone c 3, by Santa Cruz Biotechnology (2 mentions)
Anti trp1 clone ta99, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh clone 2118, by Cell Signaling Technology (2 mentions)
Imagequant las 4000 v1, by GE Healthcare (2 mentions)
Las 4000, by Cytiva (2 mentions)
Total fak, by BD (2 mentions)
Fak s910, by Thermo Fisher Scientific (2 mentions)
69 protocols using anti flag clone m2
1
Western Blot Analysis of Protein Lysates
2
Immunoblotting Analysis of Protein Extracts
3
Antibody Verification and Characterization
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The following antibodies were used in this study: anti-FLAG (clone M2, WB 1:1,000; Sigma-Aldrich), anti-GAPDH (WB 1:5,000; UBPBio), FAK S910 (WB 1:1,000, 44-596G; Invitrogen), total FAK (WB 1:1,000, 610087; BD Biosciences), and paxillin (IF 1:500; Transduction Labs). Anti-HA (WB 1:500, IP 2 μg/1 mg cell lysate, SC F-7), anti–α-tubulin (WB 1:5,000, 23948), anti-Rab40c (WB 1:500, H-8 sc514826), cul-5 (WB 1:500, H-300), and mouse ANKRD28 (WB 1:500) were purchased from Santa Cruz Biotechnology. MOB1(E1N9D) and p-MOB1(D2F10) were purchased from Cell Signaling Technology. Rabbit anti-SAPS1/2/3, ANKRD28/52, and PP6c were purchased from Bethyl Laboratories. Specificity of anti-Rab40c antibody was confirmed by immunoblotting lysates derived from cells expressing FLAG-Rab40a, FLAG-Rab40b, or FLAG-Rab40c constructs (Fig S2A ).
4
Cell Fractionation and Western Blotting
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Cell fractionation was performed as previously described (Méndez and Stillman, 2000 (link)). Western blots were done on whole-cell lysates as previously described (Bredemeyer et al., 2008 (link)). Anti–NF-κB2 (recognizes p100 and p52; polyclonal), anti-RELB (clone C1E4), anti-BLNK (clone D8R3G), anti-SYK (clone D1I5Q), anti-BTK (clone D3H5), and anti-phospho(S15)-p53 (polyclonal) antibodies were purchased from Cell Signaling Technology. Anti-TRAF2 (polyclonal), anti-TRAF3 (polyclonal), anti-IRF4 (clone E-7), anti-PU.1 (polyclonal), and anti-LaminB (clone G-1) were obtained from Santa Cruz Biotechnology. Anti-phosphorylated BLNK (clone J117-1278) was purchased from BD. Anti-GAPDH (clone GAPDH-71.1) and anti-FLAG (clone M2) were from Sigma-Aldrich. Secondary reagents were horseradish peroxidase (HRP)–conjugated anti–mouse IgG (Cell Signaling Technology) or anti–rabbit IgG (Cell Signaling Technology). Western blots were developed with ECL (Thermo Fisher Scientific) and ECL Prime (GE Healthcare).
5
Western Blot Analysis of Cellular Proteins
6
MALT1 Protease Activity Assay
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Recombinant full-length human MALT1 protein was expressed and purified43 (link). C-terminal Myc-FLAG-tagged full-length human HOIL1 was obtained from Origene. HOIL1 (0.05 μg μl−1) was incubated with different concentrations of MALT1 in assay buffer (200 mM Tris-HCl, 0.8 M Na-citrate, 0.1 mM EGTA, 0.05% CHAPS, 1 mM DTT, pH 7.4)43 (link) for 2 h at 37 °C. Cleavage of HOIL1 was analysed by 4–12% Bis-Tris SDS–polyacrylamide gel electrophoresis (PAGE) gradient gels (Life Technologies) and confirmed by immunoblotting using antibodies to N-HOIL1 (anti-N-terminal HOIL1; HPA024185; Sigma) and C-HOIL1 (anti-FLAG, clone M2, Sigma) cleavage products, respectively.
7
Immunoblot Analysis of Protein Markers
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Western blot analysis was performed following standard procedures. Bone marrow cells were directly lysed in 2X sample buffer and equal amount of the cell lysates were separated in a 10% SDS-PAGE using electrophoresis. The following antibodies were used to detect the proteins of interest: anti-FLAG (clone-M2, Sigma-Aldrich, St-Louis, USA), anti-Bmi-1 (clone-F6, Millipore Massachusetts, USA) and anti-Vinculin (Sigma-Aldrich, St-Louis, USA) antibodies. The ImageJ64 software was used to normalize the results.
8
Immunofluorescence Staining of Flag-Tagged Proteins
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Cells were fixed in 4% formaldehyde for 10 minutes on ice, permeabilised with 1% Triton-X100 for 15 minutes at RT and washed in PBS for 30 minutes. Cells were then incubated for 5 minutes in 0.2% glycine, washed for 15 minutes with 0.1% Triton-X100 in PBS for 30 minutes, blocked with 5% FCS/0.1% Triton-X100/PBS for 1 hour at RT and incubated for 1 hour with primary antibodies (anti-Flag, clone M2, Sigma; 1:4,000), washed again with 0.1% Triton-X100 in PBS for 30 minutes and incubated with secondary antibodies (anti-mouse-Alexa488, Invitrogen; diluted in blocking solution 1:100) for 1 hour. After another washing step (0.1% Triton-X100 in PBS for 30 minutes) nuclei were stained by incubating the cells with TO-PRO3 (Invitrogen; diluted 1:1,000 in PBS) for 30 minutes at RT. After washing in PBS and water cells were mounted in ProLong-Gold antifade reagent (Life Technologies) and analysed with a Leica DM IRB microscope with a TCS SP2 AOBS scanhead.
9
Protein Immunodetection and Analysis Protocol
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The following antibodies were used in this study: anti-Girdin (R&D Systems, Minneapolis, MN, #AF5345, for WB and IP), anti-4F2hc (H300, Santa Cruz Biotechnology, Santa Cruz, CA, for WB, clone MEM-108, Biolegend [#315602] for IP, and mouse monoclonal anti-4F2hc [clone HBJ 127] for IF), anti-glutathione S-transferase (GST) (Santa Cruz Biotechnology, #sc-459), anti-Myc (clone 9E10, Santa Cruz Biotechnology), anti-poly-histidine (clone HIS-1, Sigma, St. Louis, MO), anti-Flag (clone M2, Sigma), anti-β-actin (clone AC-74, Sigma), normal mouse IgG (Millipore, Milford, MA, #12–371), and normal sheep IgG (Millipore, #12–515). Antibodies to pS6K (Thr389) (#9205), S6K (#9202), S6 (#2217), pS6 (Ser240/244) (#2215), MAPK (#9102), pMAPK (Thr202/Tyr204) (#9106), Lamp1 (#9109), mTOR (#2983), and LC3B (#2775) were purchased from Cell Signaling Technology (Danvers, MA). Flag M2 affinity gel, adenosine 5′-triphosphate (ATP), and amino acids were purchased from Sigma. Phos-tag Acrylamide (Wako, Saitama, Japan) was used for the generation of the Phos-tag gel to analyze protein phosphorylation in cells.
10
Transfection and Immunofluorescence Imaging of SMAD3 in MC3T3-E1 Cells
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MC3T3-E1 cells were transfected with plasmid DNA constructs expressing SMAD3 (wild-type and p.S264Y) using X-tremeGENE HP DNA Transfection Reagent. 24 h after transfection, cells were treated with or without TGF-β1 (10 ng/ml) for 30 min and fixed with 2% paraformaldehyde for 10 min at 37°C and washed five times with 1× PBS. Cells were permeabilized with 1% BSA/0.1% Triton X-100/0.2% Saponin in 1× PBS for 40 min at room temperature and washed five times with 1× PBS before incubating at 4°C overnight with primary antibodies (anti-FLAG; clone M2, #F1804; Sigma) or anti-SMAD3 (C67H9, rabbit, #9523; Cell Signaling Technology) diluted in 2% BSA/1× PBS. Cells were washed five times with 1× PBS before blocking with 5% goat serum diluted in 1× PBS for 30 min at room temperature. Cells were then stained with secondary antibodies and nuclear dye at 4°C for 1 h protecting from light. The following secondary antibodies and fluorescent dyes were used for imaging using the IncuCyte system: Alexa Fluor 568 goat anti-mouse IgG (H+L; #A11004; Invitrogen), Alexa Fluor 488 donkey anti-rabbit IgG (H+L; #A11008; Invitrogen), and Syto61 (Invitrogen).
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