Expression and localization of BAZ1A in male germ cells.

<p>(A, B) Equal amounts of low-speed centrifuged, nuclear-enriched extracts from a panel of mouse tissues (8.5 µg protein) (A) or cultured human (U2OS and HeLa) and mouse (B16 and ear fibroblasts (EF)) cells (25 µg) (B) were immunoblotted with anti-BAZ1A, SNF2H and BAZ1B antibodies. Approximate molecular masses: BAZ1A = 178 kDa, SNF2H = 122 kDa, BAZ1B = 171 kDa and ACTIN = 42 kDa. +/+, wild-type; −/−, <i>Baz1a^−/−</i>. (C) Immunofluorescence of testis sections with BAZ1A and γH2AX antibodies. (i–iii) Testis sections at various stages (indicated by uppercase Roman numerals). Bar = 20 µm. (i′–iii′) Magnification of areas indicated in left panels. Bar = 10 µm. Arrows point to sex bodies. Note that anti-γH2AX staining along the heads of elongating spermatids at stage X-XI and the focal signal in round spermatids at stage VIII is non-specific due to cross-reactivity with the developing acrosome. ES, elongating spermatid; RS, round spermatid; Z, zygotene spermatocyte; EP, early pachytene spermatocyte; S, Sertoli cell; P, pachytene spermatocyte; L, leptotene spermatocyte; pL, preleptotene spermatocyte; M, peritubular myoid cell; D, diplotene spermatocyte. (D) Schematic summarizing BAZ1A expression during spermatogenesis, with darker shades of red indicating intensity of BAZ1A staining. Micrographs are from insets in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003945#pgen.1003945.s002" target="_blank">Figure S2B</a>; panels a–d are co-stained with HP1β, a marker of heterochromatin (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003945#pgen.1003945.s002" target="_blank">Figure S2B</a> and text for further explanation); panels e–h display higher exposures of the BAZ1A staining. RS, round spermatid; ES, elongating spermatid; CS, condensing spermatid. Abbreviations of meiotic stages as in (C). See text for full description of staining patterns. (E) Coimmunoprecipitation (IP) of SNF2H and BAZ1A from wild-type whole testis lysates. (F) Immunofluorescence of testis sections with BAZ1A and SNF2H antibodies. Magnifications of the indicated cells are shown. SP, spermatogonia; *autofluorescent red blood cells; bar = 20 µm. (G) BAZ1A immunofluorescence on squashed spermatocyte nuclei at different stages of meiotic prophase I as indicated by staining for the axial element component SYCP3. Bar = 5 µm.