Anti c myc tag 9e10 affinity gel

MutuDCs or spleen cDCs were lysed on ice in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100) supplemented with 10 mmN-ethylmaleimide (Sigma-Aldrich) and cOmplete Mini EDTA-free protease inhibitor cocktail (Roche). Post-nuclear supernatants were pre-cleared by incubation with uncoupled protein G-sepharose beads (Walter and Eliza Hall Institute Antibody Facility). MHC II was immunoprecipitated with anti-MHC II mAb (M5/114) coupled to protein G-sepharose beads (Walter and Eliza Hall Institute Antibody Facility), and Myc-tag was immunoprecipitated with anti-c-Myc-tag (9E10) affinity gel (BioLegend). Samples were eluted by denaturation in non-reducing sample buffer at 95 °C and analyzed by western blot analysis.

Whole-cell proteins were extracted from transfected HEK293T cell lines as previously described (Western blotting). A total of 3000 μg protein extract was incubated overnight at 4°C with either EGFP diluted 1:100 sc-9996 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-c-Myc Tag (9E10) Affinity Gel (BioLegend, San Diego, CA, USA), or b-actin 1:100 sc-1616R (Santa Cruz Biotechnology, Dallas, TX, USA). Antigen-antibody complexes were precipitated with protein A/G-Sepharose sc-2003 (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at 4°C and washed 3 times with cold PBSX1. Precipitated proteins were then resolved by SDS-PAGE, blotted onto nitrocellulose membranes, and reacted with the appropriate antibodies: enhanced green fluorescent protein (EGFP) diluted 1:1000 sc-9996 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-MYC diluted 1:1000 9E10 (Developmental Studies Hybridoma Bank, The University of Iowa), or anti-actin dilution 1:500 sc-1616R (Santa Cruz Biotechnology, Dallas, TX, USA).

For ubiquitination analysis, 293T cells were co-transfected with LPC-HA-Ub and pUB-TRAF3-SBP-6xHis, pUB-Myc-MFF or an empty lentiviral vector pUB-Thy1.1. At day 2 post transfection, cells were treated with 10 µM of the proteasome inhibitor MG-132 at 37°C for 4 h, and then harvested at 2 x 10⁷ cells/condition. Total cellular proteins were lysed in 1% CHAPS Lysis Buffer (39 (link)) containing 1x Phosphatase Inhibitors (Pierce) and 1 mM NEM. The insoluble pellets were removed by centrifugation at 10,000 g for 20 minutes at 4°C. The CHAPS lysates were subsequently immunoprecipitated with the Anti-c-Myc Tag (9E10) Affinity Gel (BioLegend) or Streptavidin-Sepharose beads (Pierce). Immunoprecipitates were washed 5 times with the Wash Buffer (39 (link)) containing 1x Phosphatase Inhibitors and 1 mM NEM. Ubiquitination of the immunoprecipitated MFF or TRAF3 was analyzed by immunoblot analyses.