HeLa or HEK293 cells were transfected with HA-Ub and the desired constructs. Thirty-six hours post-transfection, cells were treated with 20 μM MG132 for 6 hours. Cells were lysed in denaturing buffer (1%SDS, 50 mM Tris, pH 7.5, 0.5 mM EDTA and 1 mM dithiothreitol). After incubation at 100 °C for 10 min, the lysate was sonicated and diluted 10 times with EBC lysis buffer and incubated with anti-HA-conjugated agarose beads (Sigma, mouse antibody) for 4 hours in 4 °C. Immunoprecipitants were washed five times with NETN buffer before resolved by SDS-PAGE and immublotted with indicated antibodies. In vivo His pull down ubiquitination assays were performed as described previously 28 (link).
Anti ha conjugated agarose beads
Manufactured by Merck Group
Anti-HA-conjugated agarose beads are a laboratory product consisting of agarose beads that have been conjugated with anti-HA (hemagglutinin) antibodies. The core function of these beads is to specifically bind and isolate proteins or other molecules that have been tagged with the HA epitope, which is a commonly used protein tag for various applications in molecular biology and biochemistry.
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7 protocols using anti ha conjugated agarose beads
1
Investigating Protein Ubiquitination in Cells
2
Ubiquitin Pulldown Assay in 293T Cells
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The assay was performed as described previously [13 (link)]. Briefly, HA-tagged ubiquitin and the desired constructs were co-transfected into 293T cells. Thirty-six hours post-transfection, cells were treated with 10 μM MG132 for overnight. Cells were collected and lysed in 200 μL denaturing buffer (1% SDS, 50 mM Tris pH 7.5, 0.5 mM EDTA and 1 mM dithiothreitol). After incubation at 100 °C for 10 min, the lysate was sonicated and diluted tenfold with EBC lysis buffer and incubated with anti-HA-conjugated agarose beads (Sigma, mouse antibody) for 4 h at 4 °C. Immunoprecipitants were washed five times with NETN buffer before they were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.
3
Investigating Protein Ubiquitination in Cells
4
HA-Vpr Protein Interaction Analysis
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HeLa cells were transfected with HA- or HA-Vpr-expressing vectors using FuGENE HD (Promega, Madison, WI, USA). After 2 days incubation, the cells were lysed in CelLytic M (Sigma Aldrich), and the lysates were incubated with anti-HA-conjugated agarose beads (Sigma Aldrich) for 3 h at 4 °C. The complexes were then washed three times with Triton X-100-free wash buffer (10 mM Tris-HCl (pH 7.8), 150 mM NaCl) and analyzed by immunoblotting, as previously described [18 (link)].
5
Immunoprecipitation of Sov1-HA Fusion Protein
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A functional Sov1 fused in frame to a hemagglutinin (HA) tag was expressed from an integrative vector in strains carrying a null mutant allele of sov1. Proteins were extracted from isolated mitochondria as for sucrose gradient analysis (see above). Clarified extracts were incubated with Anti-HA-conjugated agarose beads (Sigma) or protein A-agarose beads (Thermo) as a negative control for 4 h at 4°C with gentle rocking. After centrifugation at 500 × gAV for 5 min, supernatants were recovered, and beads washed 3 times with cold phosphate-buffered saline + 0.4% digitonin. The Sov1-HA fusion protein was eluted with Laemmli buffer, and samples were analyzed by Western blotting. All antibodies used in the study are listed in Supplementary Table S5 . For Sov1-HA immunoprecipitation analysis from sucrose gradient, fractions were diluted with an equal volume of extraction buffer without digitonin prior to incubation with either Anti-HA-conjugated or protein A-conjugated agarose beads.
6
Tandem Mass Spectrometry Analysis of HA-MCL1 Interactome
7
Isolation and Detection of MED18-NRPD2a Complex
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The HA-tagged MED18 fusion construct (MED18-3xHA) under control of CaMV 35S promoter in a modified version of binary vector pCAMBIA99-1 has been previously described (Lai et al., 2014 (link)). The NRPD2a coding sequence was fused with the MYC tag (NRPD2a-6xMYC) and cloned into binary vector pBA-Myc under 35S promoter (Supplementary Table 1 ). These constructs were introduced into A. tumefaciens strain GV3101 and transformed into Arabidopsis using the floral dip method (Clough and Bent, 1998 (link)). Arabidopsis plants expressing both the MED18 and NRPD2a constructs were generated through genetic crossing. Proteins were extracted in an extraction buffer [50 mM Tris-HCl, pH7.5, 100 mM NaCl; 2 mM EDTA; 1 mM NaF; 1 mM NaVO3; 1 mM PMSF; 10 mM b-glycero phosphate; 0.1% (v/v) Triton X-100; 0.5% (v/v) Nonidet P-40; and 1x protease inhibitor cocktail]. After centrifugation, the supernatant was incubated with anti-HA-conjugated agarose beads (Sigma-Aldrich) for 12 h at 4°C. The Coimmunoprecipitation (Co-IP) products were washed with the extraction buffer four times and then detected by protein blotting.
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