For protein extraction, 5 × 105 cells per well were plated onto six-well plates and transiently transfected with indicated expression plasmids. At 48 h after transfection, cells were incubated with or without the proteasome inhibitor MG132 (10 μM) for an additional 6 h before protein extraction and western blot analysis. Primary antibodies against Flag (M2, 1:1,000) and HA (3F10, 1:4,000) were used for protein detection. For IP, HEK293 cells transfected with the indicated expression plasmids were lysed in buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 5 μg ml−1 aprotinin, 1 μg ml−1 pepstatin, 1% Nonidet P-40, 1 mM EDTA and 0.25% deoxycholate). Total cell lysates (1,000 μl) were incubated overnight with 1 μg of anti-HA or anti-Flag antibody conjugated to agarose beads (Roche Molecular Biochemicals) at 4 °C. The beads were then washed with lysis buffer, and the immunoprecipitated protein complexes were resolved by 10% SDS–PAGE. Some important original immunoblotting results are shown in Supplementary Fig. 9 .
Agarose beads
Manufactured by Roche
Sourced in United States
Agarose beads are a type of gel-based separation media commonly used in laboratory applications. They are made from agarose, a polysaccharide derived from red seaweed, and are designed to facilitate the separation and purification of various biomolecules, such as proteins, DNA, and RNA. The beads provide a porous matrix that allows for efficient size-based separation and purification of target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Protein g agarose beads, by Roche (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Torin1, by Bio-Techne (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Mounting medium with dapi, by Vector Laboratories (1 mentions)
Gp91 ds, by Bio-Synthesis (1 mentions)
Bafilomycin a, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Dithiothreitol (dtt), by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Protein a agarose, by Roche (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Ha tag, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Clean blot ip detection reagent, by Thermo Fisher Scientific (1 mentions)
Protein a agarose bead, by Roche (1 mentions)
6 protocols using agarose beads
1
Protein Extraction and Immunoprecipitation Protocol
2
HEK293T Cell Immunoprecipitation Protocol
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HEK293T cells were cultured in a 10-cm plate and transfected with the indicated plasmids for 48–72 h. Cells were then collected and lysed in immunoprecipitation (IP) buffer (1 M Tris, 0.5 M EDTA, 5 M NaCl, 100% glycerol, 100% Np40) and treated with antibodies at 4°C overnight. The lysis was then incubated with agarose beads (Roche, Indianapolis, IN, USA) for 4–6 h at 4°C. IP samples were separated by 8%–10% SDS-PAGE gels, and protein bands were detected by the chemiluminescent substrate.
3
Oxidative Stress and Autophagy Modulation
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Rotenone (RT) was purchased from Sigma-Aldrich. DCFH-DA (6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate). LysoTracKer red DND-99 was from Invitrogen. Phosphate buffered saline (PBS) was from GIBCO. The Nox-specific peptide inhibitor gp91 ds was from Biosynthesis, Lewisville, TX. LY294002 was purchased from cell signaling (9901 S). Torin1 was from Tocris Bioscience (CAS No: 1222998-36-8). Bafilomycin A was purchased from Sigma (B1793). Agarose beads were purchased from Roche. Mounting medium with DAPI was purchased from VECTASHIELD (Vector Laboratories, Inc. Burlingame, CA). WT-Src kinase and CA-Src kinase constructs were purchased from Addgene (Plasmid # 17672 and 13660 respectively). All antibodies used in this study are listed in Table S1 (Complete information about the concentration of antibodies used and companies from where we purchased).
4
Immunoprecipitation and Immunoblotting Protocol
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HeLa cells or mouse livers were lysed, centrifuged, and pre-cleared with protein G agarose beads (Roche). Supernatants were collected and 1 μg of antibody was added. After overnight incubation, 30 μl of 50% slurry of protein-G agarose beads were added, and the samples incubated at 4 °C for 2 hr. The agarose beads were pelleted by centrifugation, washed three times with ice-cold washing buffer (20 mM HEPES [pH 7.4], 0.1% NP-40, 150 mM NaCl, 0.25% sodium deoxycholic acid, 1 mM EDTA, 1 mM PMSF, 1 μM TSA, 10 mM NAM, and protease inhibitor cocktail [Roche]), resuspended in electrophoresis sample buffer (0.09 M Tris-Cl [pH 6.8], 20% glycerol, 2% SDS, 0.1 M DTT, and 0.02% bromophenol blue), boiled for 5 min, and subjected to electrophoresis, and immunoblotting.
5
Co-immunoprecipitation Assay with Modified Protocol
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Co-immunoprecipitation (Co-IP) assays were performed as described previously44 (link),45 (link) with a modified protocol using NETN buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40) containing Protease Inhibitor cocktail (Roche #11836170001) and phosphatase inhibitor cocktail (Roche #4906845001) to extract whole-cell protein, or a cell fractionation protocol for cytoplasmic and nuclear fractionation using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific #78833). Cell lysates were incubated with the indicated antibodies at 4 °C overnight. The protein complex was captured using protein A-agarose or protein G-agarose beads (Roche, Indianapolis, IN, USA) at 4 °C for 4 h, and agarose beads were collected by centrifuge and washed three times with washing buffer. The precipitated proteins were mixed with 1× Blue Loading Buffer along with 3 mM dithiothreitol (Cell Signaling Technology #7722) and subjected to western blot analysis. The following primary antibodies were used for Co-IP: BRD4 (Cell Signaling, Cat#13440, RRID: AB_2687578), HA-tag (Cell Signaling, Cat# 5017, RRID: AB_10693385), Flag-tag (Sigma-Aldrich, Cat#F1804, RRID:AB_262044), and STAT3 (Cell Signaling, Cat#9139, RRID:AB_331757).
6
RUNX1 Interactome Profiling with Small Molecules
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4 × 106 SEM cells were treated with DMSO or 10 μM of AI-4-88, AI-10-47, AI-10-104, AI-12-126 and AI-14-91 for 6 h. Cells were lysed in modified RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate and 1 mM EDTA). RUNX1 was immunoprecipitated from cell lysates using anti-RUNX1 antibody (Active Motif, cat #39000,) and protein-A Agarose beads (Roche Applied Science) as follows: cell lysates were mixed with protein A Agarose beads and 2 μg RUNX1 antibody in IP buffer I (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP40, 0.25% sodium deoxycholate) and rotated at 10 rpm for 5 h. Agarose beads were washed twice with IP Buffer I followed by washing with IP buffer II (50 mM Tris pH 7.5, 0.1% NP40, 0.05% sodium deoxycholate). All lysis, immunoprecipitation, and washing steps included DMSO/corresponding inhibitor (10 μM). The beads were heated at 95 °C for 12 min in Western blot loading buffer (100 mM Tris-HCL pH 6.8, 200 mM DTT, 4% SDS, 0.2% Bromophenol-blue, 20% glycerol). The eluted protein was resolved in a 12% polyacrylamide gel. CBFβ was detected using anti-CBFβ antibody (provided by Nancy A. Speck). The membrane was re-probed with anti-RUNX1 antibody and detected using Clean-Blot IP Detection Reagents (Thermo Scientific). Relative band intensities were quantified using ImageJ software.
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