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. 2009 Apr 24;10(6):622–628. doi: 10.1038/embor.2009.35

Figure 4.

Figure 4

Gα13 induces the ubiquitination and degradation of AhR. (A) HEK293T cells transfected as indicated were treated with or without 10 μM MG-132 for 4 h. Then, cell lysates were analysed by immunoblot (IB) analysis with the indicated antibodies. (B) HEK293T cells expressing [His]6–ubiquitin (Ub), HA–AhR and Myc-AIP with or without Gα13Q226L were lysed, and the ubiquitinated proteins were precipitated by Ni-NTA agarose. The ubiquitinated AhR, AhR and AIP in the total lysate were analysed by immunoblot. (C,D) HEK293T cells transfected as indicated were tagged metabolically with [35S]-labelled methionine and cysteine for 1 h. Then, the cells were used for pulse-chase analysis. The values of t1/2 were obtained from three independent experiments. Arrows indicate immunoprecipitated AhR. (E) Hepa1c1c7 cells were transfected as indicated and stimulated with 1 μM 3-MC for 8 h in the presence or absence of 10 μM MG-132. The expression of CYP1A1 was analysed by the quantitative RT-PCR method. 3-MC, 3-methyl cholanthrene; AhR, aryl hydrocarbon receptor; AIP, AhR-interacting protein; HA, haemagglutinin; HEK, human embryonic kidney.