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Figure 4 | Retrovirology

Figure 4

From: Nuclear matrix protein Matrin 3 is a regulator of ZAP-mediated retroviral restriction

Figure 4

Matrin 3 knockdown improves the ZAP-induced degradation of HIV-1 transcripts. Greater unspliced and multiply-spliced RNA degradation with simultaneous ZAP induction and Matrin 3 knockdown. 293TrexhZAP2 cells were transfected with control siRNA (Ctrl) or siRNA against Matrin 3 (Matrin 3 KD) for two consecutive days. The following day cells were infected with HIV-luc then treated with doxycycline treatment at 200 ng/ml (ZAP) or without. Two days postinfection, RNA was isolated from whole cell extracts (WCE), as well as from nuclear and cytoplasmic compartments. QRT-PCR analysis was performed on HIV-1 RNA using specific primers against a unspliced transcripts (Gag) and b multiply-spliced transcripts (Nef). Relative levels of these transcripts normalized to GAPDH levels are presented. Fold restriction was calculated from dividing Ctrl siRNA values by the results of each treatment indicated within the WCE, nuclear, or cytoplasmic compartment measured. Data presented are from a representative experiment done in triplicate. Similar results were obtained for three independent experiments. c QRT-PCR analysis of WCE RNA using specific primers against cellular transcripts, beta-glucuronidase (GUSB), Small Nuclear Ribonucleoprotein 70 kDa (U1), or HIV-1 Nef-luc. Fold restriction was calculated as above. Similar results were obtained for two independent experiments. d An immunoblot of the cytoplasmic (Tubulin) and nuclear fractions (Matrin 3). Compartmentalization of myc-ZAP2 is also shown.

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