Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the protein composition of the OMVs. The OMVs were mixed with SDS-PAGE sample buffer (Beyotime, Jiangsu, China) and immersed at 90 °C for 10 min. After that, the mixture was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was used for SDS-PAGE. Then, 20 µg samples were placed on a 12% (wt/vol) polyacrylamide gel. Finally, the gel was stained with Coomassie blue and imaged using a ChemiDoc system (Bio-Rad, Shanghai, China). Standard protein markers (Beyotime, Jiangsu, China) were used as molecular weight markers, ranging from 10 to 200 kDa.
Sds page sample buffer
Manufactured by Beyotime
Sourced in China, United States
SDS-PAGE sample buffer is a solution used to prepare protein samples for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The buffer contains SDS, a detergent that denatures proteins and gives them a uniform negative charge, allowing them to be separated based on their molecular weight.
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Lab products found in correlation
Polyclonal antibodies targeting ogt, by Abcam (2 mentions)
Protein g sepharose bead, by GE Healthcare (2 mentions)
Anti gfp antibody, by Beyotime (2 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Standard protein markers, by Beyotime (1 mentions)
Polyacrylamide gel, by Bio-Rad (1 mentions)
Mitochondria extraction kit, by Thermo Fisher Scientific (1 mentions)
2 5 adp sepharose beads, by GE Healthcare (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Merck Group (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Tissue or cell total protein extraction kit, by Sangon (1 mentions)
Mouse mab clone 11e7 anti cchfv pre gc, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Bioworld Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti β actin monoclonal antibody, by Cell Signaling Technology (1 mentions)
0.22 μm pvdf membrane, by Merck Group (1 mentions)
P0033, by Beyotime (1 mentions)
12 protocols using sds page sample buffer
1
SDS-PAGE Analysis of OMV Proteins
2
Characterization of ApoE-Liposome Interactions
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Recombinant human ApoE was mixed with equal amount of BSA in PBS, and incubated with sLip or SP-sLip (final concentration 5 mg mL−1 phospholipid) with different periods and rhApoE concentrations at 37 °C. The sample was centrifuged at 14k RCF for 20 min to pellet the liposome-protein complexes, which was washed with 300 μL cold PBS and transferred into a new Protein LoBind tube. The wash procedure was repeated thrice.
Proteins were desorbed from liposomes by adding SDS-PAGE sample buffer (5 × SB, Beyotime) to the pellet and boiled at 95 °C for 10 min. A 4–20% polyacrylamide gel (Biorad) was used to separate proteins. Electrophoresis was carried out at a constant current (20 mA gel−1) in electrophoresis buffer, until the dye front reached the lower end of the gel (1 h). Proteins were fixed on gel with H2O/CH3OH/CH3COOH (50:45:5, v/v/v) for 1 h under gentle agitation. Ag staining-kit (Beyotime) was used to stain gels under agitation according to the manufacturer’s manual (see Supplementary Figs.9 and 10 ).
Proteins were desorbed from liposomes by adding SDS-PAGE sample buffer (5 × SB, Beyotime) to the pellet and boiled at 95 °C for 10 min. A 4–20% polyacrylamide gel (Biorad) was used to separate proteins. Electrophoresis was carried out at a constant current (20 mA gel−1) in electrophoresis buffer, until the dye front reached the lower end of the gel (1 h). Proteins were fixed on gel with H2O/CH3OH/CH3COOH (50:45:5, v/v/v) for 1 h under gentle agitation. Ag staining-kit (Beyotime) was used to stain gels under agitation according to the manufacturer’s manual (see Supplementary Figs.
3
Immunoprecipitation of O-GlcNAc Transferase
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For immunoprecipitation, aliquots of cell homogenates were incubated with polyclonal antibodies targeting OGT (4 μg/ml) (Abcam, Cambridge, UK) at 4 °C overnight. Protein G-Sepharose beads (GE Healthcare, Chicago, IL, USA) were added to the supernatant and incubated for 3 h. The beads were washed thoroughly with lysis buffer and then eluted in SDS-PAGE sample buffer (Beyotime) by boiling for 3 min. Proteins were revealed by western blot.
4
Mitochondrial and Cytosolic Fractionation
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For preparation of cell mitochondrial and cytosolic fractions, we used a mitochondria extraction kit (ThermoFisher Scientific) according to the manufacturer's instructions. Briefly, cells were resuspended in lysis buffer and then disrupted with a Dounce homogenizer. Homogenates were centrifuged at 700 × g for 10 min at 4°C to pellet nuclei and cell debris. Supernatants were subsequently centrifuged at 12,000 × g for 15 min at 4°C, and the cytosolic fractions (supernatants) were collected. Pellets (heavy membranes enriched with mitochondria) were boiled with SDS-PAGE sample buffer (Beyotime) and analyzed by Western blotting. To determine the quality of cytosolic and mitochondrial separation, both fractions were assessed by immunoblotting for the mitochondrial marker cox-IV.
5
Western Blot Analysis of hPXR Expression
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Cells were directly lysed with 2X sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (Beyotime Institute of Biotechnology), then boiled and sonicated. The total protein was obtained following centrifugation for 10 min at 15,407 × g, then total protein was isolated, separated on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes at 100 V for 1 h. Then, the PVDF membranes were incubated with either anti-hPXR monoclonal antibody H-11 (diluted to 1:800 in blocking buffer) or anti-β-actin antibody AP0060 (diluted to 1:4000 in blocking buffer) overnight at 4°C. For staining, a goat anti-mouse HRP-labeled secondary antibody (diluted to 1:10,000 in blocking buffer) or goat anti-rabbit HRP-labeled secondary antibody (diluted to 1:2,000 in blocking buffer) were used for 1.5 h at room temperature. The protein bands were detected by an ECL detection system. Following normalization by the corresponding expression of β-actin, protein expression levels of PXR were determined by densitometry scans.
6
Immunoprecipitation and eNOS Pulldown Assay
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For immunoprecipitation, aliquots of cell homogenates were incubated with polyclonal antibodies targeting OGT (4 μg/ml) (Abcam, Cambridge, UK) overnight at 4 °C. Protein G-Sepharose beads (GE Healthcare, Chicago, IL, USA) were added to the supernatant and incubated for 3 h. The beads were washed thoroughly with lysis buffer and then eluted by boiling for 3 min in SDS-PAGE sample buffer (Beyotime).
eNOS Pull-down Assay
In BAEC and rat thoracic aorta, eNOS was extracted from the lysate by a nity precipitation using 2′,5′-ADP Sepharose beads (GE Healthcare). Cell/tissue lysates were and mixed with prepared 2′,5′-ADP Sepharose resins (50% slurry) at 4 °C for 2 h with gentle shaking. The mixture was then centrifuged at 13800 × g for 1 min. The supernatant was discarded, and the resins were washed with washing buffer (PBS supplemented with 500 mM NaCl) three times. Bound proteins were eluted by boiling the resins in 50 μl SDS-PAGE sample buffer for 10 min. For transfected cells, eNOS was extracted by a nity precipitation after transfection using the His-tag protein puri cation Kit (Beyotime) per the manufacturer's protocol.
eNOS Pull-down Assay
In BAEC and rat thoracic aorta, eNOS was extracted from the lysate by a nity precipitation using 2′,5′-ADP Sepharose beads (GE Healthcare). Cell/tissue lysates were and mixed with prepared 2′,5′-ADP Sepharose resins (50% slurry) at 4 °C for 2 h with gentle shaking. The mixture was then centrifuged at 13800 × g for 1 min. The supernatant was discarded, and the resins were washed with washing buffer (PBS supplemented with 500 mM NaCl) three times. Bound proteins were eluted by boiling the resins in 50 μl SDS-PAGE sample buffer for 10 min. For transfected cells, eNOS was extracted by a nity precipitation after transfection using the His-tag protein puri cation Kit (Beyotime) per the manufacturer's protocol.
7
Transient Protein Expression in N. benthamiana
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Agrobacterium tumefaciens-mediated transient expression in N. benthamiana leaves was performed using the agroinfiltration method as previously described (Kettles et al., 2017 (link)). Extraction of the total protein of plants or fungi was performed. Approximately 0.2 g of tissue was ground to a powder using liquid nitrogen and suspended in 1 ml of cold lysis buffer (Beyotime Biotechnology, Shanghai, China). Then, the target samples were incubated on ice for 5 min and centrifuged at 12,500 rpm for 10 min at 4°C to collect the supernatant-soluble proteins. The supernatant proteins were then mixed with 5× SDS-PAGE sample buffer (Beyotime Biotechnology, Shanghai, China) and denatured by boiling for 10 min at 100°C. The denatured proteins were separated by SDS-PAGE electrophoresis and transferred onto PVDF membranes (0.45 μm). Western blotting was carried out using an anti-GFP antibody (Beyotime Biotechnology, Shanghai, China).
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Agrobacterium tumefaciens-mediated transient expression in N. benthamiana leaves was performed using the agroinfiltration method as previously described (Kettles et al., 2017 (link)). Extraction of the total protein of plants or fungi was performed. Approximately 0.2 g of tissue was ground to a powder using liquid nitrogen and suspended in 1 ml of cold lysis buffer (Beyotime Biotechnology, Shanghai, China). Then, the target samples were incubated on ice for 5 min and centrifuged at 12,500 rpm for 10 min at 4°C to collect the supernatant-soluble proteins. The supernatant proteins were then mixed with 5× SDS-PAGE sample buffer (Beyotime Biotechnology, Shanghai, China) and denatured by boiling for 10 min at 100°C. The denatured proteins were separated by SDS-PAGE electrophoresis and transferred onto PVDF membranes (0.45 μm). Western blotting was carried out using an anti-GFP antibody (Beyotime Biotechnology, Shanghai, China).
8
Protein Extraction and Western Blot Analysis
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Total proteins from DEFs or HEK 293T cells were extracted using RIPA buffer, while total proteins from various duckling tissues were extracted using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech, Shanghai, China), according to the manufacturer's instructions. The protein samples were mixed with 5 × SDS-PAGE sample buffer (Beyotime Biotech, Beijing, China), boiled for 5 min, followed by centrifugation at 12,000 × g for 5 min at 25°C. The supernatants were subjected to electrophoresis in 10% acrylamide gels (Beyotime). For Western blot analysis, the proteins were electroblotted onto a polyvinylidene fluoride (PVDF ) membrane (Thermo Fisher Scientific, Basingstoke, UK), which was blocked with 5% nonfat milk in TBST (500 mL NaCl; 0.05% Tween 20; and 10 mM Tris-HCl, pH 7.5) for 1 h. The target proteins were probed using primary and secondary antibodies, and the PVDF membrane was visualized after incubation with a mixture of hydrogen peroxide and 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). Images were captured by the BIO-RAD ChemiDoc Imaging System (Bio-Rad, Redmond, WA). The band density was quantified using Image J Software (version 1.8.0, National Institutes of Health (NIH ), Bethesda, MD) with normalization to the β-actin signal.
9
Verifying GPLP3 Protein Display on GEM Particles
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Surface display of GPLP3 proteins on GEM particles (G-GP) was verified by SDS-PAGE, Western Blot, and the immuno-electron microscope (IEM).
For the SDS-PAGE and WB identification of the binding GEM particles, the GPBLP3 was resuspended in 5 × SDS-PAGE sample buffer (Beyotime Biotechnology, Shanghai, China), separated by 10% SDS-PAGE and then transferred by electroblotting onto NC transfer membranes under denaturing conditions for Western blotting with a mouse anti-CCHFV-GN specific for CCHFV-GN polyclonal antibody (diluted 1:500), a mouse mAb clone 11E7 anti-CCHFV pre-Gc (diluted 1:1000; BEI Resources, Manassas, VA, USA), a mouse mAb clone 11E7 anti-CCHFV pre-Gc (diluted 1:1000, BEI Resources, USA), and an HRP-conjugated secondary antibody (diluted 1:5 000; Bioworld, Minnesota, MN, USA).
For IEM, a mouse antiserum specific for CCHF-GN (diluted 1:200), mouse mAb clone 11E7 anti-CCHFV pre-Gc (diluted 1:1000), a mouse mAb clone 11E7 anti-CCHFV pre-Gc (diluted 1:1000), the immuno-electron microscope anti-Mouse IgG (whole molecule) Gold antibody produced in goat (diluted 1:200, Sigma, St. Louis, MO, USA) were used. Samples were observed under the electronic microscope.
For the SDS-PAGE and WB identification of the binding GEM particles, the GPBLP3 was resuspended in 5 × SDS-PAGE sample buffer (Beyotime Biotechnology, Shanghai, China), separated by 10% SDS-PAGE and then transferred by electroblotting onto NC transfer membranes under denaturing conditions for Western blotting with a mouse anti-CCHFV-GN specific for CCHFV-GN polyclonal antibody (diluted 1:500), a mouse mAb clone 11E7 anti-CCHFV pre-Gc (diluted 1:1000; BEI Resources, Manassas, VA, USA), a mouse mAb clone 11E7 anti-CCHFV pre-Gc (diluted 1:1000, BEI Resources, USA), and an HRP-conjugated secondary antibody (diluted 1:5 000; Bioworld, Minnesota, MN, USA).
For IEM, a mouse antiserum specific for CCHF-GN (diluted 1:200), mouse mAb clone 11E7 anti-CCHFV pre-Gc (diluted 1:1000), a mouse mAb clone 11E7 anti-CCHFV pre-Gc (diluted 1:1000), the immuno-electron microscope anti-Mouse IgG (whole molecule) Gold antibody produced in goat (diluted 1:200, Sigma, St. Louis, MO, USA) were used. Samples were observed under the electronic microscope.
10
Confirmation of duTRIM32 Protein Expression
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Western blotting analyses were performed to confirm pcDNA3.0-duTRIM32-Flag expression. After 48 h after transfection with expression plasmid or empty vector, the DEF were lysed with a RIPA lysis buffer (P0013C; Beyotime, Shanghai, China) containing 1 mM PMSF (ST506, Beyotime). Lysates were centrifuged at 12,000 rpm for 5 min at 4°C, the supernatant was collected, and its protein concentration was determined. After adding 5 × SDS-PAGE sample buffer (Beyotime), samples were boiled for 10 min and run immediately on 10% SDS-PAGE gels. Protein samples were transferred to 0.22 μm PVDF membrane (Millipore) using NcmBlot Rapid Transfer Buffer (WB4600; New Cell & Molecular Biotech, suzhou, China). The PVDF membrane was blocked with NcmBlot blocking buffer (P30500; New Cell & Molecular Biotech) for 10 min at room temperature and then incubated with mouse anti-Flag monoclonal antibody (8146; Cell Signaling Technology, Shanghai, China) or anti–β-actin monoclonal antibody (3700; Cell Signaling Technology) overnight at 4°C. The membrane was then incubated with Peroxidase AffiniPure Goat Anti-Mouse IgG, the secondary antibody (33201ES60; Yeasen), for 1 h at room temperature. After each incubation, the membrane was washed thrice for 10 min each with 1x TBST. The protein bands were visualized with an NcmECL Ultra kit (P10100; New Cell & Molecular Biotech).
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