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Figure 2 | Molecular Cancer

Figure 2

From: Leucine-rich repeat protein PRAME: expression, potential functions and clinical implications for leukaemia

Figure 2

PRAME LRR repeats, subcellular localisation and interaction with nuclear receptors. A: Predicted domain structure of the human PRAME sequence indicating potential Leucine Rich Repeats (LRRs). The LRRs are numbered and indicated by the blue arrows; residues conserved in typical LRRs are highlighted in bold. The black boxes indicate regions predicted to have a high probability of α-helicity, and two predicted NLS sequences are underlined. The boxed area in red is a region implicated in interaction with retinoic acid receptors, and potentially contains LXXLL and CoRNR box-like motifs. B: Subcellular localisation of endogenous PRAME proteins in leukaemia cell lines. Leukaemia cell lines were cultured as described in the legend to Fig. 1, and harvested onto coverslips using a cytospin centrifuge. Cells were fixed in 4% paraformaldehyde, permalised with 0.2% Triton X-100 and blocked with 3% PBS prior to application of an α-PRAME antibody (Abcam ab31285) followed by secondary antibody (Alexa Fluor 594 chicken anti-rabbit IgG - Invitrogen A21442). DNA was stained with a Hoechst stain (Sigma Aldrich 332581). Images were captured using LSM 510 Meta confocal laser scanning microscope (Zeiss). C: Yeast two-hybrid experiments to assess interactions of SRC1 nuclear receptor interaction domain (431-761) or full-length PRAME (1-509) with nuclear receptors were performed using the reporter strain S.cerevisiae L40 as described previously [68, 69]. PRAME and SRC1 domains were expressed as LexA fusion proteins. Nuclear receptor ligand binding domains (RARα 200-464; RXRα 230-467; ERα 282-595; AR 625-919) were expressed as VP16 activation domain (411-490) fusion proteins, and reporter (β-galactosidase) specific activity was determined as described previously [68, 69].

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