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. 2012 Dec 26;7(12):e52827. doi: 10.1371/journal.pone.0052827

Figure 1. LUNA deletion mutants do not disrupt pp71.

Figure 1

A) Sequence of the start codon region for UL8-82ast in FIX-WT and FIX-ΔLUNA viruses. The upper strand represents the coding frame for UL82 and its translation, the bottom strand represents the coding frame for UL81-82ast and its translation. Red-labeled nucleotides and amino acids indicate those that changed after mutagenesis. B) Hind III digest of FIX-BAC-WT (lane 1), FIX-BAC-(GalK-KAN)-A (lane 2), FIX-BAC-ΔLUNA (lane 3), FIX-BAC-(GalK-KAN)-B (lane 4) and FIX-BAC-Rev (lane 5). FIX-BAC-(GalK-KAN)-A contains the Galk-Kan insert before the mutation; FIX-BAC-(GalK-KAN)-B is the re-introduction of the insert in the process of creating the revertant. C) Southern blot of the digest in (B) with a GalK specific probe. The Galk-Kan insert is indicated by the arrow. D) HF cells were infected with FIX-WT, FIX-ΔLUNA or FIX-Rev respectively over a 20day time course. The presence of the pp71 protein was detected via western blotting. 20 µg of protein was added per well, samples were loaded in the following order for each time point: FIX-WT, FIX-ΔLUNA and FIX-Rev. Lane 1: Mock HF, lanes 2–4: HF 1 dpi, lanes 5–7: HF 3 dpi, lanes 8–10: HF 5 dpi, lanes 11–13: HF 10 dpi, lanes 14–16: HF 20 dpi. Blots were incubated with primary antibodies goat anti-pp71 (1∶500) and mouse anti-actin (1∶10,000) and secondary antibodies anti-goat or anti-mouse IgG HRP (1∶1000).