XB-IMG-78870
Xenbase Image ID: 78870
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Figure 2. EGFP-Rpl10a protein localizes properly and can be used to selectively enrich for RNAs. A: Western analysis of whole eye lysates from transgenic tadpoles expressing cytoplasmic GFP (GFP-cyto) and EGFP-Rpl10a, both under control of the same islet2b regulatory sequence, and from wildtype control tadpoles (Wt) show transgene protein products of the expected size. B: Representative image shows a 2D projection of a series of optical sections (Z-stack) showing nucleolar and inner segment localization of the EGFP-Rpl10a protein in rods. C: Representative image shows a 2D projection of a Z-stack showing nucleolar and cytoplasmic localization of the transgenic EGFP-Rpl10a protein in RGCs. Arrowheads in B and C point to nucleoli. D: RNA recovery from unconjugated beads (−) or EGFP-coated beads (+) using lysates of Tg(isl2b:EGFP-Rpl10a) (L10a), Tg(isl2b:GFP) (GFP-cyto), and from non-transgenic wildtype (Wt) retinas, show significant RNA recovery only in Tg(isl2b:EGFP-Rpl10a) lysates in the presence of the EGFP antibody. Grey horizontal line shows the 0.5-ng limit of detection of the bioanalyzer. E: The residual unbound RNA fraction from the samples shown in D was comparable in all samples. Data in D and E represent the mean RNA levels from 12 retinas averaged for three replicates, and statistical significance between individual samples was established by pair-wise ANOVA comparisons (***P < 0.001). F: Representative superimposed electropherogram traces from TRAP EGFP-rpl10a transgenic samples affinity purified with EGFP conjugated beads (red trace) and unconjugated beads (black trace) show high abundance 18S and 28S ribosomal RNAs and lower abundance mRNAs only in EGFP-rpl10a samples extracted using EGFP- conjugated beads. Right insets show corresponding RNA gels for each of the traces. Scale bars = 2 μm.Download figure to PowerPoint Image published in: Watson FL et al. (2012) Copyright © 2012. Image reproduced with permission of the Publisher, John Wiley & Sons. Larger Image Printer Friendly View |