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. Author manuscript; available in PMC: 2012 Oct 15.
Published in final edited form as: Oncogene. 2007 Sep 10;27(10):1412–1420. doi: 10.1038/sj.onc.1210784

Figure 3.

Figure 3

E4-ORF1 monomers bind and sequester MUPP1, MAGI-1 and ZO-2. (a) Monomeric AAA binds MAGI-1. 293 T cells were co-transfected with pGW1 encoding HA-MAGI-1 and either pGW1 (vector) or pGW1 encoding wt or the indicated mutant E4-ORF1. Extracts in RIPA buffer were IP’d with HA antibody. Recovered proteins and total cell extracts (Input) were blotted with HA or E4-ORF1 antibody. E4-ORF1 PBM mutant IIIA (see Figure 2a) is a negative control. (b) Monomeric AAA sequesters endogenous MUPP1 and MAGI-1. CREF cells were transduced with pBABE (vector) or pBABE encoding wt or the indicated mutant E4-ORF1. Extracts in RIPA buffer were separated into soluble supernatant (S) and insoluble pellet (I) fractions. Equivalent amounts of each fraction were blotted with MUPP1, MAGI-1, ZO-2, SAP97 (Dlg1) or E4-ORF1 antibody. (c–e) E4-ORF1 monomers bind PDZ domains of MUPP1, MAGI-1 and ZO-2. 293 T cells were transfected with pGW1 encoding wt E4-ORF1 and/or pGW1 encoding (c) HA-MUPP1 PDZ10, (d) HA-MAGI-1 PDZ1, or (e) HA-ZO-2 PDZ1. Extracts in RIPA buffer were fractionated by SEC with elution buffer lacking DOC and blotted with E4-ORF1 or HA antibody.