Journal: PLoS Genetics
Article Title: The Broad-Spectrum Antiviral Protein ZAP Restricts Human Retrotransposition
Figure Lengend Snippet: ZAP inhibits exogenous L1 RNP levels in cells. (A) Expression of HA-ZAP-L, HA-ZAP-S, and V5-TEV-MOV10 reduces the amount of ORF1 protein (top IP panel) and ORF2p RT activity (bottom IP panel) in α-FLAG antibody purified pc-L1-1FH immunoprecipitates. RT activity was determined by the LEAP assay [ 81 ]. Below: Coomasie-stained gel of cell lysates prior to IP showed no obvious loss of global protein levels in the presence of ZAP. (B) Analysis of 293T cell lysates showing that ORF1 protein from pc-L1-1FH is reduced in the presence of ZAP and MOV10, but not empty vector or an unrelated protein, C22ORF28 (top panel). Panels below show by Western blotting that neither ZAP nor MOV10 alter expression of endogenous HSP90 or GFP protein expressed from cotransfected CEP-EGFP. Lysates were prepared from two pooled wells of a six-well plate. (C) Expression of ZAP causes loss of L1 RNA. Top panels: RT-PCR of L1 RNA expressed from 99-PUR-JM111-EGFP, which expresses L1-RP with an ORF1 mutation that prevents genomic insertion. PCR primers spanned the intron of the EGFP reporter cassette to distinguish spliced RNA products from contaminating plasmid DNA (generating a 105 nt band vs a 1 kb band). PCR reactions are shown in the presence or absence of RT. Bottom panels: Levels of endogenous HSPA6 RNA were the same in the presence or absence of ZAP.
Article Snippet: Subsequent PCR used GoTaq DNA polymerase (Promega).
Techniques: Expressing, Activity Assay, Purification, Staining, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Polymerase Chain Reaction