To assess endogenous EGFR ubiquitination, NRCMs were infected with adenovirus siRNA-control or siRNA-UCHL1 for 24 hours. To assess ligand-induced EGFR ubiquitination, NRCMs infected with siRNA-control or siRNA-UCHL1 were incubated with EGF (20 ng/ml; Thermo Fisher Scientific, Waltham, MA, USA) and treated with MG132 6 hours before harvest. EGFR was immunoprecipitated with anti-EGFR and detected by Western blot analysis with antibody against Ub. To examine overexpressed EGFR ubiquitylation, HEK293T cells were transfected with expression vectors for GFP-tagged EGFR, Myc-tagged UCHL1 (WT or C90S mutant), or Flag-tagged ubiquitin as indicated in Fig. 4 using Lipofectamine 2000 (Thermo Fisher Scientific). Lysate proteins from cultured cells and heart tissues were precipitated and analyzed by Western blotting using appropriate antibodies (including EGFR, Ub, GFP, Flag, and Myc) as described previously (40).

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