4.8. Ubiquitination Assay

Whole-cell lysates were extracted after treating cells for 4 h with the proteasome inhibitor MG132 (10 mM). Non-specific binding was eliminated by first incubating cell lysates with protein A/G (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h, after which lysates were incubated with anti- TGFBR antibody (Abcam, Biotech, Life Science; ab124894) overnight at 4 °C. The next day, lysates were incubated with protein A/G for 1 h at 4 °C. Ubiquitinated TGFBR signals were detected with an anti-ubiquitin antibody (Epitomics Inc., Burlingame, CA, USA).