HeLa cells transfected with indicated plasmids were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature. Cells were then blocked with 3% BSA in PBS for 1 hr and incubated with appropriate combinations of mouse anti-FLAG (1:100; Clontech Laboratories) and rabbit anti-HA (1:3,000; CST) antibodies diluted in blocking solution for 1 hr at room temperature. This was followed by proximity ligation assay using the Du-olink detection reagent kit (Olink Bioscience) according to the manufacturer’s protocol. Fluorescence images were acquired using an Olympus Fluoview FV1200 confocal microscope with a 60×/1.35 UPlanSApo objective. Images were prepared and analyzed with Fiji software (http://fiji.sc ). For each experiment, signals from at least 50 cells were quantified. All data were analyzed using GraphPad PRISM software.
Mouse anti flag
Manufactured by Takara Bio
Mouse anti-FLAG is a monoclonal antibody that binds specifically to the FLAG epitope tag. It is commonly used for the detection and purification of recombinant proteins expressed with a FLAG tag.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Prism software, by GraphPad (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Duolink detection reagent kit, by Olink (2 mentions)
Fluoview fv1200 confocal microscope, by Olympus (2 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Guinea pig anti synaptophysin, by Synaptic Systems (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
4 protocols using mouse anti flag
1
Proximity Ligation Assay for Protein-Protein Interactions
2
Proximity Ligation Assay for Protein-Protein Interactions
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HeLa cells transfected with indicated plasmids were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature. Cells were then blocked with 3% BSA in PBS for 1 hr and incubated with appropriate combinations of mouse anti-FLAG (1:100; Clontech Laboratories) and rabbit anti-HA (1:3,000; CST) antibodies diluted in blocking solution for 1 hr at room temperature. This was followed by proximity ligation assay using the Du-olink detection reagent kit (Olink Bioscience) according to the manufacturer’s protocol. Fluorescence images were acquired using an Olympus Fluoview FV1200 confocal microscope with a 60×/1.35 UPlanSApo objective. Images were prepared and analyzed with Fiji software (http://fiji.sc ). For each experiment, signals from at least 50 cells were quantified. All data were analyzed using GraphPad PRISM software.
3
Immunofluorescence and Western Blot Antibodies
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The following antibodies were used for immunofluorescence: mouse anti-neurofilament (clone 2H3, Developmental Studies Hybridoma Bank), guinea pig anti-synaptophysin (Synaptic Systems). The following antibodies were used for western blot: rabbit anti-α tubulin (ab18251; Abcam), rabbit anti-GFP (GTX113617; Genetex), mouse anti-GFP (12A6; Developmental Studies Hybridoma Bank), mouse anti-GFP (9F9.F9; Novus Biologicals) mouse anti-FLAG (clone M2, F3165; Sigma), mouse anti-FLAG (635691; Clontech), mouse anti-β-actin (A1978; Sigma), mouse anti-α tubulin (T6199; Sigma). For visualization of AChR we used fluorescently labeled α-bungarotoxin (B35451 or B13422; Thermo), and for visualizing the synaptic cleft, we used fasciculin II, which binds to acetylcholinesterase (F-225; Alomone Labs, labeled with FluoReporter FITC kit from Thermo).
4
Immunoprecipitation of PAR3 and ApoER2
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IFRS1 cells (2 × 10 6 ) were transfected by electroporation with DNA constructs encoding Tiam1-CA1199-HA (Meseke et al., 2013b) and PAR3 or ApoER2-Full-length-FLAG, Rap and PAR3 (10 μg in total). After 48 h of expression, cells were washed twice in ice-cold PBS and lysed in ice-cold immunoprecipitation buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1.0% Triton X-100, 10 mM EDTA and protease inhibitors). Specific antibodies (Rabbit-PAR3 (Millipore 07-330) or mouse-HA) (Fuentealba et al., 2007) and Protein A-Sepharose beads were incubated for 4 h at 4 °C. After washing, the beads-antibody complex was incubated with the lysates for 2 h. Finally, the samples were suspended in sample buffer, resolved by SDS-PAGE, and transferred to Immobilon PVDF membrane (Millipore). The co-precipitating levels of proteins were visualized by Western blotting using a mouse anti-FLAG (Clontech) or Anti-PAR3 antibodies.
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