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. 2005 Jan 3;168(1):89–101. doi: 10.1083/jcb.200408155

Figure 2.

Figure 2.

Sequence requirements for MLV L-domain activity and interaction with HECT ubiquitin ligases. (A) Virion production by 293T cells transfected with MLV proviral plasmids bearing the indicated mutations in the DPPPYR sequence. Virion and cell lysates were analyzed by Western blotting with αMLV Gag antibodies. (B) Yeast two-hybrid analysis of interactions between the indicated mutant MLV Gag proteins and HECT ubiquitin ligases. The mean level of β-galactosidase expression in yeast transformed with the indicated Gal4 and VP16 Gag and ubiquitin ligase fusion proteins is shown. Error bars indicate the SD from the mean of triplicate measurements. (C) Binding of endogenous WWP1, WWP2, and Itch to wild-type and mutant GST-p12 proteins expressed in 293T cells. The top panels show Western blots of the glutathione-bound proteins, probed with the indicated specific antibodies and the bottom panel shows Coomassie blue staining of the glutathione-bound GST-p12 proteins, verifying equivalent expression and loading.