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. 2010 Jul 20;30(18):4391–4403. doi: 10.1128/MCB.00002-10

FIG. 4.

FIG. 4.

SPO11β polypeptide is expressed in early prophase I whereas SPO11α is only expressed once spermatocytes advance past early pachynema. (A) Generation of a monoclonal antibody (Ab) that can immunoprecipitate (Ip)/immunodetect SPO11 in mouse testis extracts. Total testis extracts (6 to 10 mg of total protein) from wild-type (wt) and Spo11−/− mice were immunoprecipitated with anti-SPO11 (or nonspecific rabbit [R] IgG) and subsequently analyzed by Western blotting with the same antibody, anti-SPO11. M, molecular weight marker. (B) Presence of SPO11 in anti-SPO11 immunoprecipitates confirmed by mass spectrometry. Five peptides mapping to different areas of the protein (including exon 2 [blue]) identified SPO11 with 100% certainty in a wild-type extract immunoprecipitated with anti-SPO11 antibody (see Materials and Methods). The relatively low coverage (13%) did not allow identification of specific isoforms, given that a coverage above 95% would be required to identify SPO11α through the absence of peptides mapping to exon 2. (C) The estimated molecular masses of the proteins detected by the SPO11 antibody are in the range expected for SPO11 isoforms. Estimated molecular masses of the four bands detected by the antibody and expected molecular masses for the SPO11 isoforms annotated in Ensembl. (D) Testes from wild-type juvenile mice containing spermatocytes in early stages of prophase I contain primarily the SPO11β polypeptide. Testis extracts from juvenile mice at 11 dpp (early prophase I) and Dmc1−/− and wild-type adult mice were immunoprecipitated/blotted with anti-SPO11 antibody. (E) Mutants arrested in midprophase express primarily the SPO11β polypeptide, whereas a mutant arrested in metaphase I expresses both isoforms. Total testis extracts from wild-type, Spo11−/−, Dmc1−/−, Hop2−/−, Atm−/−, and Mlh1−/− mice (5 to 10 mg of total protein) were precipitated/blotted with anti-SPO11 antibody. Mutant testes are 0.2 to 0.5 the size of wild-type testes, so 3 or 4 mutant mice were used in order to immunoprecipitate extracts containing comparable amounts of total protein. The table specifies the number of mice used for each sample, the milligrams of total protein used for the Ip, and the α/β ratios. (F) Amount of SPO11 isoform precipitated from each extract normalized to total protein and to the wild-type extract. Shown is a comparison of the amounts of SPO11 isoform precipitated from mutant and wild-type extracts, taking into consideration the difference in total protein between extracts. For each isoform, the band intensity was divided by the amount of total protein in the extract and this ratio was divided by the corresponding ratio obtained for the wt sample. AU, arbitrary units.