The DNA plasmids mEmerald-ATG5-C-18 (Addgene plasmid #54000) or mCherry-ATG5-C-18 (Addgene plasmid #54995) were mixed with the BioT transfection reagent (Bioland) in serum-free DMEM to a final concentration of 2 μg/mL per the manufacturer’s protocol. This transfection mixture was incubated at room temperature for 20 minutes prior to inoculation into cells. Two days after transfection, cells were harvested for further studies. For infection studies, Huh7.5 cells were infected with HCV using a multiplicity of infection (m.o.i.) of 1 one day after transfection. Infected cells were then harvested one day post-infection for further analysis. All of our infection studies were conducted using a variant of the HCV JFH1 isolate, which replicated more efficiently than the original JFH1 isolate [41 (link)].
Memerald atg5 c 18
Manufactured by Addgene
MEmerald-ATG5-C-18 is a plasmid that encodes the ATG5 protein fused to the mEmerald fluorescent protein. ATG5 is a key component of the autophagy pathway.
Automatically generated - may contain errors
2 protocols using memerald atg5 c 18
1
Transfection and HCV Infection Assay
2
Plasmid constructs for HBV protein expression
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The plasmid p1.3xHBV contains the overlength 1.3mer HBV genomic DNA in the pUC19 vector (51 (link)). p1.3xHBV PC– (PC MT), and p1.3xHBV S– (S MT) contained 1.3mer HBV genome with mutations that abolished the expression of the HBV precore protein and HBsAg, respectively. The S MT was constructed by mutating nucleotide 223 from T to A to generate a premature termination codon. This mutation did not affect the overlapping polymerase coding sequence. HBV protein expression plasmids, including pCMV-precore, pCMV-core, CM1, and CM2 had been described before (36 (link)). The plasmid pcDNA-p22 was constructed by deleting the signal peptide-coding sequence (amino acids 2 to 19) from pCMV-precore. The DNA plasmid mEmerald-ATG5-C-18 was purchased from Addgene (Addgene plasmid #54000). siRNA targeting human Rab11a (SASI_Hs01_00126206) and a negative control siRNA (SIC001) were purchased from Sigma-Aldrich. The DNA and siRNA transfections were performed using Lipofectamine 3000 (Invitrogen) and TransIT-X2 (Mirus) per the manufacturer’s instructions.
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