Journal: Journal of Virology

Article Title: Transcription Factor YY1 and Its Associated Acetyltransferases CBP and p300 Interact with Hepatitis Delta Antigens and Modulate Hepatitis Delta Virus RNA Replication

doi: 10.1128/jvi.02581-07

Figure Lengend Snippet: FIG. 4. YY1 forms a large complex with HDAgs in vivo. (A) The nuclear extracts (500 g) of the HDAg-producing (L10 or S7) and the parental HepG2 (G2) cells were isolated and subjected to sucrose gradient centrifugation analysis. Aliquots of each fraction were analyzed by immuno- blotting using antibodies against YY1 or HDAg. Protein standards, catalase (232 kDa) and thyroglobulin (669 kDa), were run in parallel experiments, and their sedimented positions are indicated. (B) Complex formation between HDAgs, YY1, CBP, and p300. The sucrose gradient fractions 15 to 17 obtained from nuclear extracts of HepG2 (G2), L10, or S7 cells as described in panel A were pooled together and immunoprecipitated (IP) with human anti-HDAg antiserum. The immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with antibodies against HDAg, YY1, CBP, and p300. Input, nuclear extracts (50 g) of G2, L10, or S7 as loading controls; , anti.

Article Snippet: The immunoprecipitates were washed with washing buffer (PBS containing 0.3% NP-40) and processed for immunoblotting analysis with antibodies against YY1 (C-20; Santa Cruz), CBP (C-20; Santa Cruz), and p300 (N-15; Santa Cruz).

Techniques: In Vivo, Isolation, Gradient Centrifugation, Immunoprecipitation, SDS Page, Western Blot

Journal: Journal of Virology

Article Title: Transcription Factor YY1 and Its Associated Acetyltransferases CBP and p300 Interact with Hepatitis Delta Antigens and Modulate Hepatitis Delta Virus RNA Replication

doi: 10.1128/jvi.02581-07

Figure Lengend Snippet: FIG. 6. CBP and p300 modulate the HDV replication. (A) CBP and p300 enhance SHDAg expression. For each experiment, approximately 2 106 HuH-7 cells were transfected with 10 g of empty vector () or expression construct of FLAG-tagged CBP or Myc-tagged p300 by Lipofectamine 2000 transfection reagent (Invitrogen). At 12 h posttransfection, cells were then transfected with HDV genomic RNA (15 g) and SHDAg mRNA (5 g) by DMRIE-C transfection reagent (Invitrogen). The expression levels of CBP, p300 (1 day posttransfection), and HDAg (4 days posttransfection) were analyzed by SDS-PAGE followed by immunoblotting with anti-FLAG tag, anti-Myc tag, or anti-HDAg antibodies. (B) CBP and p300 enhance the replication of HDV RNA. The expression levels of HDV genomic (G) and antigenomic (AG) RNA in the transfected cells as described in panel A were determined by Northern blotting at 4 days posttransfection. (C) Acetyltransferase-defective mutant of p300 inhibits HDV replication. This experiment is similar to that shown in panel A, except that 10 g of empty vector () or expression construct of Myc-tagged p300 (p300) or its mutant D1399Y (D1399Y) was used for the transfection experiment. At 12 h posttransfection, cells were then transfected with HDV genomic RNA (10 g) and SHDAg mRNA (10 g) by DMRIE-C transfection reagent (Invitrogen). The expression levels of p300 and HDAg (1 day posttransfection) were analyzed by SDS-PAGE followed by immunoblotting with anti-Myc tag or anti-HDAg antibodies at 24 h posttransfection. (D) The expression levels of HDV RNAs and SHDAg in the transfected cells as described in panel C were determined by Northern blotting and Western blotting at 4 days posttransfection. Mock, HuH-7 cells without transfection. P, SHDAg or HDV RNAs as positive controls; WB, Western blotting; , anti.

Article Snippet: The immunoprecipitates were washed with washing buffer (PBS containing 0.3% NP-40) and processed for immunoblotting analysis with antibodies against YY1 (C-20; Santa Cruz), CBP (C-20; Santa Cruz), and p300 (N-15; Santa Cruz).

Techniques: Expressing, Transfection, Plasmid Preparation, Construct, SDS Page, Western Blot, FLAG-tag, Northern Blot, Mutagenesis