In order to examine the localization of the expressed CrCAO protein, immunogold labeling was used with transmission electron microscopy. Harvested cells of WT and NsChlb19 transformant were washed with PBS buffer before fixation in ice for 20 min with fixation agent (4% paraformaldehyde with 0.1% glutaraldehyde). After washing three times with PBS buffer, cells were harvested in PBS solution containing 1% gelatin. Then, washing step after centrifugation for 2 min at 3000 rpm was repeated, increasing the content of gelatin in PBS from 2.5%, 7.5% and to 10%. For the steps with 2.5% gelatin and 7.5% gelatin, cells were incubated at 37 °C for 10 min. In the last step with 10% gelatin, cells were incubated in ice for 20 min. The samples were then sliced into 0.5–1 mm, which were supplemented with 2.3 M sucrose at 4 °C for overnight. After slicing the sample into sections, primary antibody of anti-FLAG-tag antibody (Cell Signaling Technology, USA) was used, followed by secondary gold-labeled anti-rabbit IgG antibody (Abcam, UK). Transmission microscopy image was taken with Tecnai G2 spirit TWIN transmission microscope (FEI, USA).
Anti flag tag antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-flag tag antibody is a laboratory reagent used for the detection and purification of proteins that have been engineered to contain a specific amino acid sequence known as the 'flag tag'. This antibody binds to the flag tag, allowing researchers to identify and isolate the tagged proteins from complex mixtures.
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Lab products found in correlation
Anti notch3 antibody, by Cell Signaling Technology (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
R960 25, by Thermo Fisher Scientific (1 mentions)
A21203, by Thermo Fisher Scientific (1 mentions)
Anti v5 tag antibody, by Thermo Fisher Scientific (1 mentions)
H 1500, by Vector Laboratories (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Dm2500, by Leica (1 mentions)
Viromerred transfection reagent, by BioNTech (1 mentions)
Endofree plasmid purification kit, by Qiagen (1 mentions)
Pmaxgfp plasmid, by Lonza (1 mentions)
Magna rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Pierce rna 3 end desthiobiotinylation kit, by Thermo Fisher Scientific (1 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
β actin, by ZSGB-BIO (1 mentions)
Plvx ires puro vector, by Takara Bio (1 mentions)
Puromycin, by Merck Group (1 mentions)
Pvdf membrane, by Proteintech (1 mentions)
Enhanced chemiluminescence detection kit, by Proteintech (1 mentions)
9 protocols using anti flag tag antibody
1
Immunogold Localization of CrCAO Protein
2
Immunohistochemical Staining of Flag-Tagged Proteins
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The tumor tissue sections were incubated with 70 μl 10% BSA at 37 °C for 40 min to block nonspecific binding sites, then incubated at 4 °C with anti-Flag Tag Antibody (Cell Signaling Technology, #2368) overnight, and then incubated with secondary antibody for 40 min at 37 °C. A a fresh amount of DAB chromogenic agent (50 μl DAB concentrate in 1 ml DAB substrate solution) was added at room temperature for 5–10 min.
3
Visualizing Recombinant PCSK6 and Corin in Cardiomyocytes
4
Generating SIRT6 Mutant Plasmids
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Plasmid pcDNA3.1 containing an insert to express human SIRT6 (Addgene 13817) was used as the template for site-directed mutagenesis. Cysteine at position 144 was mutated to serine by introducing a single base exchange using the QuikChange site-directed mutagenesis kit. Mutants were confirmed by DNA sequencing. Plasmids were isolated using Endofree plasmid purification Kit (Qiagen).
For some experiments THP-1 cells were transfected with 1 microgram plasmid using either GeneX Plus transfection reagent (American Type Culture Collection) or Viromer Red transfection reagent (Lipocalyx) according to manufacturer’s instructions. For other experiments THP-1 cells were transfected with 500 nanograms plasmid using CellLine V nucleofector kit (Amaxa) according to manufacturer’s instructions. pMax GFP plasmid (Lonza) was used as a control plasmid to visualize transfection efficiency. Overexpression of SIRT6 protein was confirmed by immunoblotting transfected cell lysates with anti-FLAG tag antibody (Cell Signaling).
For some experiments THP-1 cells were transfected with 1 microgram plasmid using either GeneX Plus transfection reagent (American Type Culture Collection) or Viromer Red transfection reagent (Lipocalyx) according to manufacturer’s instructions. For other experiments THP-1 cells were transfected with 500 nanograms plasmid using CellLine V nucleofector kit (Amaxa) according to manufacturer’s instructions. pMax GFP plasmid (Lonza) was used as a control plasmid to visualize transfection efficiency. Overexpression of SIRT6 protein was confirmed by immunoblotting transfected cell lysates with anti-FLAG tag antibody (Cell Signaling).
5
RNA-protein Interaction Profiling
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RNA was transcribed in vitro using a MEGAscript T7 Transcription Kit (Invitrogen) and biotinylated using a Pierce RNA 3’ End Desthiobiotinylation Kit (Thermo Scientific) according to the manufacturers’ instructions. Cells were prepared using Pierce IP lysis buffer (Thermo Scientific). RNA pull-down assays were performed with a Pierce Magnetic RNA–Protein Pull-Down Kit. According to the manufacturer’s instructions, biotinylated RNA was captured with streptavidin magnetic beads and then incubated with cell lysates or purified protein (20 µg) at 4 °C for 6 h before washing and elution of the RBP complex. The eluted proteins were subjected to MS analysis or western blotting. RIP assays were performed with a Magna RNA-binding protein immunoprecipitation kit (Millipore, Bedford, MA) according to the manufacturer’s instructions. Negative control IgG, human anti-PFKFB3 antibody (1:20, Abcam, ab181861) and anti-FLAG tag antibody (1:20, Cell Signaling Technology, 8146) were used in this study. After proteinase K digestion, the immunoprecipitated RNAs were extracted, purified, and subjected to qPCR. RNA levels were normalized to the input (10%).
6
Detecting scFv Expression in Nude Mice Organs
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The expression of scFv in the organs of the nude mice was detected by Western blot. Briefly, the nude tissue was ground in a mortar, and lysed in RIPA buffer with PMSF for 30 min to extract all tissue proteins. The protein solution was run on an SDS–PAGE and transferred to polyvinylidene fluoride (PVDF). The PVDF membrane was incubated at room temperature with the anti-Flag Tag Antibody (Cell Signaling Technology, #2368) at 1:1000, and then with secondary antibody at a 1:1000 dilution. β-actin (ZSGB-BIO, Beijing, China,#TA-09) was used as an internal control.
7
Generating GLUT1-Flag Expressing Cell Lines
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Complementary DNA (cDNA) for human glucose transporter 1 (GLUT1) was amplified from Raji Burkitt’s lymphoma cells with a PCR reaction adding sequence encoding a flag tag to the 3’ end of the gene in frame with GLUT1. Next, the GLUT1-flag gene was cloned into the pLVX-IRES-Puro vector (Clontech Laboratories, Inc, Mountain View, California, USA) for subsequent lentiviral expression and verified with DNA sequencing. HEK293T cells were cotransfected with pLVX-GLUT1-flag-IRES-Puro or pLVX-IRES-Puro (for controls), envelope (pMD2.G), and packaging (psPAX2) vectors using standard calcium phosphate protocol. pMD2.G and psPAX2 plasmids were obtained from professor Didier Trono (École Polytechnique Fédérale de Lausanne, Switzerland). Lentivirus-containing supernatants were collected 48 h post transfection, centrifuged overnight at 4°C at 3000×g, and added to the culture of HEK293T cells for 24 h. Mixture of positive clones was selected with puromycin (Sigma) and evaluated with Western blotting using anti-flag tag antibody (Cell Signaling, cat #2368) as described below. Two independent sets of GLUT1-flag-expressing cells and empty vector-expressing cells were obtained for further studies.
8
Protein Extraction and Western Blot Analysis
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To obtain total cellular protein, cells were lysed in RIPA buffer (Beyotime) containing 0.5 mM PMSF (Beyotime) and 1x phosphatase inhibitor (Roche). To separate cytoplasmic and nuclear protein, Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) was applied according to the instructions. The protein samples were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). The PVDF membranes were blocked in 5% fat-free milk for 2 h, followed by interactions with primary antibodies overnight at 4 . Next, the PVDF membranes were incubated with second antibodies for another 2 h and interacted with Enhanced Chemiluminescence Detection Kit (Proteintech). The grey levels were analyzed by using ImageJ software. The total and nuclear protein levels were normalized by β-actin and LaminB1, respectively. The following antibodies were used: anti-β-actin antibody (Proteintech, 66009-1-Ig, 1:5000), anti-LaminB1 antibody (Proteintech, 66095-1-Ig, 1:5000), anti-flag tag antibody (Cell signaling technology, #14793, 1:1000), anti-NOTCH3 antibody (Cell signaling technology, #5276, 1:1000), anti-NF-κB p65 antibody (Wanleibio, WL01980, 1:500), HRP Goat Anti-Mouse IgG (Jackson, 115-035-003, 1:10000), HRP Goat Anti-Rabbit IgG (Jackson, 111-035-003, 1:10000).
9
Immunoprecipitation of NOTCH3 Protein
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In brief, cells were lysed in IP buffer (Beyotime) containing 0.5 mM PMSF (Beyotime) and 1x phosphatase inhibitor (Roche). Cell lysate was centrifuged and supernatants are precleared with 10 μl protein A/G beads (Beyotime) at 4˚C for 1 h. Cell lysate was then incubated with antibodies at 4˚C overnight prior to addition of 20 μl protein A/G beads for 2 h at 4˚C. Immunoprecipitated proteins were extensively washed and heat denatured at 95˚C for 5 min. The protein samples were separated by SDS-PAGE and analyzed by Western blot. The following antibodies were used: anti-flag tag antibody (Cell signaling technology, #14793, 1:50), anti-NOTCH3 antibody (Cell signaling technology, #5276, 1:200), Normal IgG (Sigma-Aldrich, 12-371, 1:1000).
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