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. 2010 Oct 7;6(10):e1001150. doi: 10.1371/journal.pgen.1001150

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Doucet et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A. Cellular localization of the L1-encoded proteins: Immunofluorescence was conducted on pAD2TE1 transfected U-2 OS cells 48 hours post-transfection. T7-tagged ORF1p (green; left column) and TAP-tagged ORF2p (red; middle column) staining are shown for representative transfected cells. A merged image is shown in the rightmost column; DAPI (grey) was used to stain nuclear DNA. Cartoons of the constructs are indicated at the left of the micrographs. The blue rectangle in the constructs indicates the mneoI cassette. B. Cellular localization of L1-encoded proteins and RNA: Immunofluorescence/RNA FISH was conducted on pAD3TE1 transfected U-2 OS cells 48 hours post-transfection. T7-tagged ORF1p (green), TAP-tagged ORF2p (blue), L1 RNA (red), and DAPI (turquoise) staining are indicated in left four micrographs. A merged image is shown in the rightmost panel. The cartoon of pAD3TE1 is shown above the micrographs. C. L1 cytoplasmic foci are associated with stress granules: Immunofluorescence/fluorescence microscopy was performed on U-2 OS cells co-transfected with pAD2TE1 and one of the following plasmids: 1) pAgo2-GFP (green staining, top row of images); 2) pDCP1á-GFP (green staining, second row of images); 3) pG3BP-GFP (green staining, third row of images). T7-tagged ORF1p (red), and TAP-tagged ORF2p (blue) also are shown. A merged image is shown in the rightmost panels; DAPI (grey) was used to stain nuclear DNA. Immunofluorescence also was performed on U-2 OS cells transfected with pAD3TE1. Images using antibodies against the endogenous stress granule components eIF3 (áeIF3 (green)) and G3BP (áG3BP (green)) are shown. L1 RNA (red), and ORF2p (blue) also are indicated. Arrows indicate the association of L1 cytoplasmic foci (white) and stress granules (yellow). A merged image is shown in the rightmost columns; DAPI (grey) was used to stain nuclear DNA. All L1 constructs in panel B and C contain the mneoI retrotransposition indicator cassette.