Total, cytoplasmic and nuclear extracts were prepared as previously described [13 (link), 28 (link), 29 (link)]. For immunoprecipitation (IP) experiments, nuclear extracts were resuspended in dilution buffer (50 mM TRIS pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 0.5% NP40, protease and phosphatase inhibitors). Dynabeads magnetic beads (Thermo Fisher Scientific) were incubated for 1 h at room temperature on a rotating wheel with the indicated antibodies. Nuclear extracts were then added to the beads and the incubation was continued for 2 additional hours. Immuno-complexes were purified by using a magnet (DynaMag-2, Thermo Fisher Scientific), the beads were washed five times with dilution buffer and eluted in 4X lithium dodecyl sulphate (LDS) sample buffer (Thermo Fisher Scientific) for WB analysis. Detailed information for all antibodies is provided in Supplementary Table S1 . Total RNA was isolated from cells using EuroGOLD TriFast reagent (Euroclone) according to the manufacturer’s instructions. The amount of RNA was measured by using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific).
Dynabeads magnetic beads
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Dynabeads are superparamagnetic beads designed for a variety of applications in research and diagnostics. They feature a uniform size and shape, and can be functionalized with various ligands to capture and isolate target molecules or cells from complex samples.
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Lab products found in correlation
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Ago2 antibody, by Merck Group (2 mentions)
Proteinase k, by Roche (2 mentions)
Complete lysis m, by Roche (2 mentions)
Dynabeads protein g immunoprecipitation kit, by Thermo Fisher Scientific (2 mentions)
Anti ubiquitin, by Santa Cruz Biotechnology (2 mentions)
Facsaria 2, by BD (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Dynamag 2, by Thermo Fisher Scientific (1 mentions)
Eurogold trifast reagent, by Euroclone (1 mentions)
Cd4 cd8 microbeads, by Miltenyi Biotec (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Abi 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Ht exo sap it, by Thermo Fisher Scientific (1 mentions)
Exosome human cd63 isolation detection kit, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Control sirna, by Bioneer (1 mentions)
Tnf α elisa kit, by R&D Systems (1 mentions)
38 protocols using dynabeads magnetic beads
1
Comprehensive Cellular Fractionation and Analysis
2
On-Bead Hybridization Capture of BST-DSN Probes
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At first, capture using BST–DSN probes generated from PCR products was examined employing an on-bead hybridization capture procedure as described by Maricic et al. (5 (link)). Briefly, 800 ng of BST–DSN probes generated from a mix of 10 PCR products were denatured at 98°C for 2 min and immobilized on Dynabeads™ magnetic beads (Thermo Fisher Scientific) at room temperature for 15 min. Immobilized beads were then washed three times with 1× BWT (1 M of NaCl, 5 mM of Tris–Cl, pH8.0, 0.5 mM of ethylenediaminetetraacetic acid (EDTA), pH8.0) and 0.05% of Tween-20 buffer and re-suspended with 500 ng denatured LMPCR products in 1× oligonucleotide buffer, following by incubation at 65°C for 16 h. After washing one time with BWT buffer and two times with 1× Phusion buffer, the captured DNA was released in 1× Phusion buffer by incubation at 95°C for 2 min. Target-specific capture was then validated by two-step PCR (Supplementary Figure S1 ).
3
Enrichment and Activation of Antigen-Specific T Cells
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T cells were positively enriched using LS columns and CD4/CD8 MicroBeads according to the manufacturer’s protocol (Miltenyi Biotec). Isolated T cells were cultured in serum-free or serum-containing media in the presence or absence of cytokines. Subsequently, T cells were stimulated with either Expamers or Dynabeads Magnetic Beads (Thermo Fisher Scientific) according to in-house S.O.P. or manufacturer’s recommendations, respectively. Activated T cells were kept in culture for at least 7 days unless indicated otherwise.
For antigen-specific activation, Strep-tagged pMHCs were produced as described (Knabel et al., 2002). To generate pMHC/CD28 Expamers, Streptagged pMHC (HLA-A*02:01/pp65495-503) and anti-CD28 Fab fragment were added to the Strep-Tactin multimer backbone and complexed for 15 min at RT. Subsequently, pMHC/CD28 Expamers were added to PBMCs of an HLA-A*02:01+ CMV seropositive donor. Cells were checked before and after 8 days in vitro cell culture for the presence of HLA-A*02:01/pp65495-503 specific T cells by flow cytometry staining with fluorescently labeled pMHC multimer and anti-CD8 (PE; clone HIT8a) antibody.
For antigen-specific activation, Strep-tagged pMHCs were produced as described (Knabel et al., 2002). To generate pMHC/CD28 Expamers, Streptagged pMHC (HLA-A*02:01/pp65495-503) and anti-CD28 Fab fragment were added to the Strep-Tactin multimer backbone and complexed for 15 min at RT. Subsequently, pMHC/CD28 Expamers were added to PBMCs of an HLA-A*02:01+ CMV seropositive donor. Cells were checked before and after 8 days in vitro cell culture for the presence of HLA-A*02:01/pp65495-503 specific T cells by flow cytometry staining with fluorescently labeled pMHC multimer and anti-CD8 (PE; clone HIT8a) antibody.
4
Quantitative Immunoprecipitation and Western Blot Analysis of Autophagy and Lipid Metabolism Proteins
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Protein expression (Atg7, PLIN-1, p62, CETP, and LCAT) was analyzed using protein extracts (60–125 mg for immunoprecipitation) from liver homogenates that were isolated using Dynabeads Magnetic Beads (Thermo-Fisher Scientific, MA, USA), and visualized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), where bands were detected by chemiluminescent reaction. Signal density was quantified by densitometry using ImageJ software: Atg7, PLIN-1, p62/SQSTM-1, CETP, and LCAT were analyzed using immunoprecipitation, while LC3B and Beta-actin antibodies purchased from Cell Signaling Technology, CA were analyzed from Western blots (diluted 1:1,000, while 10 μg of antibody was used for IP).
5
Chmp2b Lysine 6 Methylation Analysis
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For identification of CHMP2B lysine 6 methylation (K6) on endogenous CHMP2B protein, HeLa cells were lysed in lysis buffer (20 mM Tris HCl, pH 8, 150 mM NaCl, 0,4% to 1% NP40, 2 mM EDTA, protease inhibitors) and CHMP2B was immunoprecipitated (IP) with CHMP2B antibody and Dynabeads magnetic beads (Thermo Fisher Scientific). IP product was loaded on SDS PAGE gel and CHMP2B band was excised and in-gel digested by using chymotrypsine (Promega). For label Label Free Quantification (LFQ) analysis of CHMP2B lysine 6 (K6) methylation, HeLa cells were co-transfected with GFP CHMP2B and flag SMYD2 or flag SMYD2 Y240A (dead catalytic mutant). Twenty-four hours post transfection, HeLa cells were lysed in lysis buffer (20 mM Tris HCl, pH 8, 150 mM NaCl, 0.4% to 1% NP40, 2 mM EDTA, protease inhibitors) and GFP CHMP2B was IP with GFP antibody and Dynabeads magnetic beads. The IP product was loaded on SDS PAGE gel and the GFP CHMP2B band was excised and in-gel digested by using chymotrypsine. Peptide extracted from each band are vacuum concentrated to dryness and resuspended in loading buffer (0.3% TFA in miliQ water) before nanoLC-MS/MS analysis.
6
SARS-CoV-2 Variant Identification Protocol
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The purification of PCR amplification products was performed with HT Exo SAP-IT (Thermo Fisher Scientific). The sequencing reaction was carried out using the BigDye™ Terminator V.3.1 Cycle Sequencing kit (Applied Biosystems). Sequencing products were then purified using Invitrogen™ Dynabeads™ Magnetic Beads (ThermoFisher Scientific). The purified products were analyzed on the ABI 3500 Genetic Analyzer (Applied Biosystems). Raw sequences data were edited, aligned and assembled using the BioEdit Sequence Alignment Editor, Version 7.2. Delta, Omicron BA.1 and Omicron BA.2 variants were identified according to the presence of specific mutations as described in Fig. 1 .
7
ChIP Assay of FoxO1 in Mouse Liver
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ChIP assays using mouse liver was performed as described previously (Nakagawa et al., 2016 (link); Takeuchi et al., 2016 (link)). Briefly, liver tissue samples from the fasted and re-fed mice were minced in PBS and cross-linked in 1.5% formaldehyde for 15 min at room temperature. Fixed samples were homogenized and then subjected to sonication for DNA fragmentation. After centrifugation, supernatant was diluted and then subjected to immunoprecipitation with anti-FoxO1 or with control IgG bound to Dynabeads magnetic beads (Thermofisher) and rotated overnight at 4°C. The complexes were washed and then incubated overnight at 65°C for reverse crosslinking. DNA-protein complex was treated with Proteinase K and chromatin DNA was purified with phenol-chloroform, eluted in TE buffer and subjected to Q-PCR analysis. The primer sets are listed in Table S3 .
8
Co-immunoprecipitation of SNF2H Protein
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For co-IP, protein G Dynabeads magnetic beads (Thermo Fisher Scientific, 10004D) were washed twice and resuspended in their original volume with RIPA buffer diluted (1:1) in dilution buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT and protease inhibitor). Nuclear extracts for the IP were obtained, as described in the previous section. For preclearing, 15 µl of beads were added to nuclear extracts and incubated for 30 min at 4 °C with overhead rotation. Five percent of the precleared lysate was used as input control. To the rest of the precleared lysates, 5 μg of anti-SNF2H antibody was added and samples were left overnight at 4 °C with rotation. The following day, 25 µl of prewashed magnetic beads were added and samples were left for 1 h at 4 °C with rotation. After this time, beads were washed three times with 500 µl IP-wash buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl 2 , 0.5% NP-40, 1 mM DTT and protease inhibitor). Beads were then transferred to a new tube and washed again twice before being resuspended in an appropriate volume of gel-loading buffer (1:1 mix of RIPA buffer and 5× Laemmli buffer with 5% β-mercaptoethanol) for SDS-PAGE and western blotting. The antibodies used for co-IP and western blotting are reported in Supplementary Table 6.
9
Flow Cytometric Detection of MkExos
10
Detailed Reagents and Materials Protocol
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TSA, brefeldin A (BFA), glyburide, probenecid, suberoylanilide hydroxamic acid (SAHA), valproic acid, butyrate and lipopolysaccharide (LPS) were purchased from Sigma Aldrich (St. Louis, MO, USA). Lipofectamine® RNAiMAX, Opti-MEM™ media, and Dynabeads® Magnetic beads were purchased from Thermo Fisher Scientific (San Jose, CA, USA). ABCA1 small interfering RNA (siRNA), control siRNA, and primers were purchased from Bioneer (Daejeon, Korea). Complementary DNA synthesis kit and PCR PreMix kit were purchased from Intron biotechnology Inc. (Seongnam, Gyeonggi-do, Korea). Mouse tumor necrosis factor (TNF)-α ELISA kit was purchased from R&D system (Minneapolis, MN, USA). Anti-N-cadherin antibody was purchased from Abcam (cat.no. ab18203, Cambridge, MA, USA); anti-β-actin antibody was purchased from Sigma Aldrich (cat.no. A5316, St. Louis, MO, USA); Monoclonal anti-ABCA1 antibody (cat.no. sc-58219, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and polyclonal antibody (cat.no. PA1-16789, Invitrogen, Waltham, MA, USA) were used. Monoclonal (cat.no. NB100-116, Novus Biologicals, Littleton, CO, USA) and polyclonal antibodies against APE1/Ref-1 (cat.no. MR-PAAPE, MediRedox, Daejeon, Korea) were used.
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