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RhCMV inhibits HC expression in the absence of RhUS2-11.

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posted on 2008-10-03, 00:07 authored by Colin J. Powers, Klaus Früh

All experiments were performed at 24 hours post infection at MOI = 3. A) Immunoblot analysis of MHC-I or calreticulin in Mock- or RhCMV-infected TRF lysates. B) IP of total MHC-I upon labeling with 35S-Met/Cys for the indicated time. (*) All IPs from WT and recombinant RhCMV- infected cells contain antibody-binding proteins around 55kDa (see Fig. S2) which likely correspond to the RhCMV homologues of the Fc-receptor UL119-118 of HCMV [50]. Since these viral proteins are not involved in MHC-I inhibition they are not shown in most figures. C) Pulse-chase labeling of 10 min and immunoprecipitation of total MHC-I from Mock-infected, HCMV-infected THFs, or RhCMV-infected TRFs. D) Pulse-labeling of 60 min and IP of MHC-I, Tfn Rec (Transferrin receptor) or Vimentin from Mock-infected or RhCMV-infected TRFs. E) Pulse-chase labeling of 10 min and IP of total MHC-I or HC. Cells were labeled as in 1C, but lysed in SDS buffer prior to IP. F) Pulse-chase labeling and IP of RhCMV-infected TRFs treated with proteasome inhibitor. Where indicated TRFs were incubated with 50 µM MG132 or DMSO during 60-min of Met/Cys starvation, 10-min label, and 30-min chase. For control, TRFs were transduced with AdUS11 (MOI = 25), a recombinant adenovirus expressing HCMV US11, for 24 hours followed by NP40-lysis and IP with K455. Shown for RhCMV-infection is both NP-40 lysis (top panel) and SDS-lysis (bottom panel) prior to IP with the noted antibody.

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