Plasmids for HBV expression HBV-producing plasmids were isolated

Plasmids for HBV expression. HBV-producing plasmids were isolated and constructed as previously but described (26). A partial HBV genome sequence (approximately 3,056 bp) which contains the complete HBV envelope open reading frame was amplified using primers B2600 NheI (5��-CCG CTA GCC TTA CAG TAA ATG AAA A-3��) and B25R (5��-TCC CAC CTT ATG TGT CCA-3��) via PCR and then cloned into the NheI/HindIII sites of vector plasmid pcDNA3.1(?) (Invitrogen, San Diego, CA). Another partial HBV genome sequence (approximately 1,859 bp) was amplified with primers B980 NheI (5��-GGA AAG TAT GCT AGC GAA TTG TGG-3��) and B2839 BstEII (5��-CCA AGA ATA TGG TGA CCC-3��) by PCR and then cloned into the NheI/BstEII sites of the plasmid containing the previously described 3-kb partial HBV sequence to become a replicative-form HBV construct.

In all HBV recombinant plasmids, a cytomegalovirus promoter drives the transcription of the pregenomic RNA. Culture and transfection of Huh-7 cells. The well-differentiated HCC cell line Huh-7 was used (33). The cells (106) were transfected with FuGENE HD transfection reagent (Roche, Inc.) according to the supplier’s instructions. For analysis of snail, twist, vimentin, and E-cadherin expression, 15 to 30 ��g of L-HDAg- or S-HDAg-expressing plasmid DNA was transfected into Huh-7 cells and the medium was changed at 6 h posttransfection. Subsequently, the medium was replaced and collected on days 3, 6, and 9 as previously described (26). Western blot analysis.

The isolated proteins were separated by SDS-PAGE, blotted onto nitrocellulose membranes, and stained for HDAgs with anti-HDV-positive human serum (1:5,000), for HBsAgs with monoclonal antibody A10F1 (1:2,000), or for the reference protein heat shock cognate 70 (Hsc70) with monoclonal antibody HSC70 (B-6; 1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA) (26). For analysis of the expression of snail, twist, vimentin, and E-cadherin, cell lysates were harvested on days 6 and 9 posttransfection. The membranes were probed with a primary antibody incubated at 4��C overnight, followed by the addition of a secondary antibody. Snail was detected by using rabbit anti-snail antibody (C15D3; 1:1,000; Cell Signaling Technology, Inc.), twist was detected by using rabbit anti-twist antibody (ab50581; 1:1,000; Abcam, Cambridge, MA), E-cadherin was detected by using rabbit anti-E-cadherin antibody (4065; 1:1,000; Cell Signaling Technology, Inc.

), and vimentin was detected by using mouse anti-vimentin antibody (V6630; 1:1,000; Sigma-Aldrich). The relative quantitative values of EMT markers were estimated by using the AlphaImager 2000 Documentation Analysis System and the AlphaImager 2000 software Anacetrapib package. Northern blot analysis. A total of 20 ��g of RNA was analyzed by Northern blotting as previously described (26).

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