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. 2011 Mar 7;108(12):4870–4875. doi: 10.1073/pnas.1101273108

Fig. 2.

Fig. 2.

Itch promotes polyubiquitination and subsequent degradation of LATS1 by 26S proteasome. (A) In vitro ubiquitination of LATS1 by Itch. Immunoprecipitated LATS1-myc on beads was used as a substrate in an ubiquitination assay with a ligase buffer containing E1, E2, Ubiquitin-FLAG, ATP, and Itch-GST or Itch-C830A-GST. After reaction, the beads containing LATS1-myc were washed extensively with modified RIPA buffer, followed by Western blot analysis using anti-FLAG antibody. (B) In vivo ubiquitination of LATS1 by Itch. Ubiquitin-HA and different combination of Itch-Myc, Itch-ligase-dead mutant (Itch-C830A-Myc), and LATS1-FLAG were transfected into COS7 cells. Ubiquitinated LATS1 was detected by immunoprecipitation of LATS1 with anti-FLAG antibody, followed by detection of ubiquitin using anti-HA antibody. (C) Proteasome inhibitor blocks Itch-induced LATS1 degradation. COS7 cells transfected with either LATS1-FLAG alone or LATS1-FLAG together with Itch-Myc were treated with either DMSO (control) or proteasome inhbitor (MG132). (D) Lysosome inhibitor fails to block Itch-induced LATS1 degradation. COS7 cells transfected with either LATS1-FLAG alone or LATS1-FLAG together with Itch-Myc were treated with either DMSO (control) or lysosome inhibitor (Baf A1).