The ubiquitination assay was performed by incubating 3.0 µg of a bacterially expressed GST-β-catenin, GST-MyHCemb fragment (1041–1941 a.a.) and GST-Alix with 150 ng (1.2 μM for each 10 μl reaction) of purified recombinant E1 (UBE1-BostonBiochem), 200 ng UbcH5b (11.7 μM for each 10 μl reaction), 1.0 µg (~ 6.4 μM for each 10 μl reaction) CRL5Ozz ubiquitin ligase and 7.5 µg (781.2 μM for each 10 μl reaction) of ubiquitin or ubiquitin K48 mutant (BostonBiochem) in a final volume of 30 µl of ubiquitination buffer (0.05 M Tris–HCl, pH 7.6; 0.01 M MgCl2, 0.004 M ATP) for 60 min at 30 °C.
To analyze the ubiquitinated products, the ubiquitination reactions were diluted in 500 μl RIPA buffer (50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% deoxycholate, 0.1% SDS, 1 mM EDTA, protease inhibitors and phosphatase inhibitors), immunoprecipitated with 5 µl anti-β-catenin (BD-Bioscience), 20 µl anti-MyHCemb (2B6) or 20 µl anti-Alix (d’Azzo Lab), resolved on 7.5% SDS–polyacrylamide gel, and immunoblotted with anti-ubiquitin (ThermoFisher Scientific), anti-β-catenin, anti-MyHCemb or anti-Alix antibodies.
To analyze the ubiquitinated products, the ubiquitination reactions were diluted in 500 μl RIPA buffer (50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% deoxycholate, 0.1% SDS, 1 mM EDTA, protease inhibitors and phosphatase inhibitors), immunoprecipitated with 5 µl anti-β-catenin (BD-Bioscience), 20 µl anti-MyHCemb (2B6) or 20 µl anti-Alix (d’Azzo Lab), resolved on 7.5% SDS–polyacrylamide gel, and immunoblotted with anti-ubiquitin (ThermoFisher Scientific), anti-β-catenin, anti-MyHCemb or anti-Alix antibodies.