Transcription factors are usually unstable proteins. The degradation of the majority of transcription factors is through the ubiquitin proteasome pathway and is tightly regulated by E3 ubiquitin ligases. KLF5 is an important transcription factor regulating cell proliferation, cell cycle, survival, migration, differentiation, angiogenesis, and stem cell self-renewal. We have shown that the WWP1 E3 ligase targets KLF5 for ubiquitin-mediated degradation. Several methods to determine whether a protein is ubiquitinated have been described [Kaiser, Tagwerker (Methods Enzymol 399:243–248, 2005); Bloom, Pagano (Methods Enzymol 399:249–266, 2005)]. This chapter focuses on experimental approaches testing KLF5 transcription factor ubiquitination and degradation by its E3s.
