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Figure 2 | PathoGenetics

Figure 2

From: Mutations in the nuclear localization sequence of the Aristaless related homeobox; sequestration of mutant ARX with IPO13 disrupts normal subcellular distribution of the transcription factor and retards cell division

Figure 2

Missense mutations in nuclear localization sequence (NLS) regions of the Aristaless related homeobox (ARX) homeodomain disrupt the normal nuclear subcellular localization in HEK293T cells. (A) Schematic of the human ARX protein. Known functional domains are highlighted in the open reading frame: Octapeptide (OP) as horizontally hatched rectangle, NLS as three black rectangles, polyalanine tracts (PA) as four white rectangles, acidic domain as vertically hatched rectangle, homeodomain crosshatched and Aristaless domain (OAR) hatched. The sequences flanking the homeodomain are shown above the black rectangle, the locations indicated at either end, with the basic residues that are part of the NLS-like motifs in bold. (B) Individual focal planes of cell transfected with Myc-R379L (NLS3) mutation construct show aggregates of mutant ARX protein form both inside the nucleus and outside in the cytoplasm surrounding the nucleus. Panel on far left is at the top of the cells (focal plane 2) with subsequent images an additional four focal planes further through the cell, maximal projection of all images is shown on the far right. The aggregates often displace the nuclear material, shown by distortion and absence of the blue DAPI signal in location of the aggregates. Scale bar = 10 μM. (C) Representative pictomicrographs of the localization of the Wt and mutant ARX protein 24 hrs post transfection. My~ ARX detected by anti-ARX Ab and Cy3 conjugated secondary antibody (left panel) merged with DAPI stained nuclear material (right panel). Scale bar = 10 μM. (D) The percentage of transfected cells displaying abnormal localization as inclusions or aggregates in the nucleus, with or without aggregates in the cytoplasm, was determined from between 1000 and 4000 transfected cells per construct, from at least two separate transfection reactions, 24 h post-transfection. Full-length Myc-tagged constructs transfected are listed along the bottom of the graph; ARX Wt (open bar), R332P (light grey bar), T333N (grey bar), P353L mutant (cross hatched bar), R379L and R379S (both dark grey bars) and double mutant R332P-R379L (black bar). All groups comparison of the percentage of transfected cells with abnormal subcellular localization of expressed ARX protein was achieved by parametric Kruskal-Wallis test. * P < 0.05 versus ARX-Wt, # versus ARX-NLS mutants with single aa substitutions.

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