MEFs were transfected with various plasmid pairs (listed in Supplemental Table S3) in a 1:1 ratio using jetPRIME (Polyplus Transfections) transfection reagent according to the manufacturer's instructions. Twenty-four hours after transfection, cells were treated with 10 µM proteasome inhibitor MG-132 (Sigma-Aldrich) for 4 h. Subsequently, protein isolation was carried out using cell lysis buffer (150 mM NaCl, 50 mM Tris-HCl at pH 8.0, 1% Triton X-100) supplemented with protease inhibitor (Roche) (30-min incubation at 4°C followed by two freeze–thaw cycles). From each lysate, an aliquot was saved for input analysis by Western blot while the remaining lysate was incubated with the appropriate antibody (Supplemental Table S6B) on a rotating wheel overnight at 4°C. The next day, 20 µL of Dynabeads Protein G for immunoprecipitation (ThermoFisher Scientific) was added to each lysate followed by a 2-h incubation on a rotating wheel at 4°C. The samples were washed twice with cell lysis buffer before the immunoprecipitates were eluted from the beads with NuPAGE LDS sample buffer (Invitrogen). Samples were boiled for 10 min at 98°C and then loaded on 4%–12% Bis-Tris NuPAGE gels (Thermo Fisher). Western blotting was carried out as described above with appropriate primary antibodies and HRP-linked rabbit anti-mouse IgG (light chain-specific [CSL]) secondary antibody followed by chemiluminescent detection. Antibodies used for immunoprecipitation are listed in Supplemental Table S6B.

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