XB-IMG-126097
Xenbase Image ID: 126097
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Fig. 3. Subcellular localization of somatic XLAP2 protein in adult tissues and XTC cultured cells. a, b Immuno-gold staining of ultra-thin sections of embedded tissues with antibodies against XLAP2, followed by incubation with protein A conjugated to 8-nm gold particles (Cyt cytoplasm, NE nuclear envelope, Nuc nucleus). In brain (a) and muscle (b) tissues, XLAP2 (black dots) localizes mostly at the inner nuclear membrane (arrows) and peripheral heterochromatin (arrowheads) but is also present in intra-nuclear regions (double arrowheads). b Note that XLAP2 occurs both in the nucleus of the myotube (top) and in the muscle satellite cell (bottom). Bar 500 nm. c In early prophase of XTC cells, costaining for XLAP2 (green) and phospho-histone H3 (red) reveals that XLAP2β is located not only at the NE, but also in intra-nuclear loci (arrows). Bar 5 μm. d Confocal studies of interphase XTC tissue cultured cells stained for XLAP2 demonstrate that XLAP2β is present in NE and in invaginations of the nuclear membrane (strong dots inside the nucleus). Note that the region lying on the upper left side of the nucleus corresponds to the nucleolus area (see merge). Bar 10 μm. e Biochemical properties of XLAP2β protein from XTC tissue-cultured cells. Analysis of the XLAP2β polypeptide solubility in isolated cell nuclei, extracted with 10 mM TRIS buffer alone or containing Triton X-100 (TX), NaCl, Triton X-100 plus NaCl (TX+NaCl), or urea (6M Urea). Equivalent amounts of the supernatants and pellets were resolved in SDS-PAGE gel, transferred onto nitrocellulose filter and probed for XLAP2 protein. XLAP2-66 displayed the properties of an integral membrane protein involved in interactions with other protein components of the nuclear lamina, because it was solubilized in the presence of Triton X-100 together with NaCl. f–j Transmission electron microscopy (TEM) studies of XTC tissue-cultured cells confirm the NE localization for XLAP2 proteins. XTC cells were grown in culture, embedded in gelatin and cryo-sectioned for immuno-gold TEM. XLAP2 was detected with antibodies against XLAP2 followed by Protein A coupled with 5-nm gold particles. Gold-labelled XLAP2 was mostly localized at the nuclear periphery and inner nuclear membrane (INM, arrowheads) but intra-nuclear clusters (double arrowheads) were also present (f, j). g Higher magnification of the boxed area in f (Cyt cytosol, ONM outer nuclear membranes, Nuc nucleus). Bars 100 nm Image published in: Chmielewska M et al. (2011) Image downloaded from an Open Access article in PubMed Central. © The Author(s) 2011 Larger Image Printer Friendly View |