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. Author manuscript; available in PMC: 2017 Jul 24.
Published in final edited form as: Discov Med. 2013 Jan;15(80):7–15.

Figure 2.

Figure 2

Identification of β-TRCP and IKKβ as upstream negative regulators of Twist. A. β-TRCP consensus phospho-degron motif and an amino acid sequence of human Snail β-TRCP degron. B. Alignment of evolutionarily conserved putative β-TRCP degron motifs at the N-terminal amino acid sequence of Twist1. C. HeLa cells were infected with the indicated lentiviral shRNA constructs for 24 hours. Uninfected cells were eliminated by selection with puromycin for 48 hours. Equal amounts of whole cell lysates were immunoblotted with the indicated antibodies. D. Immunoblot analysis of whole cell lysates and immunoprecipitates derived from HeLa cells transfected with hemagglutinin (HA)-Twist and the indicated Flag-β-TRCP1 constructs. After 30 hours posttransfection, cells were pretreated with 15 μM MG132 for 10 hours to block the proteasome pathway before harvest. E. Immunoblot analysis of whole cell lysates derived from HeLa cells transfected with the indicated constructs.