JCS02362 Supplementary Material

Supplemental material

Generation of antibodies against mouse proteins SYCP1, SYCP2, SMC1b, REC8 and STAG3. To analyse the distribution of cohesins in female germ cells during meiosis, we generated polyclonal antisera against peptides derived from each of the three mouse meiosis-specific cohesion proteins (SMC1b, REC8 and STAG3) and the SC proteins SYCP1 and SYCP2. The affinity-purified antibodies against SYCP1, SYCP2, STAG3 and SMC1b recognize single protein bands on western blots with molecular weights corresponding to those previously reported (Prieto et al., 2001; Revenkova et al., 2001). The anti-REC8 antibody recognizes a series of 80-90 kDa bands in testes, in accordance with previous reports (Eijpe et al., 2003; Lee et al., 2003). The SYCP1, SYCP2, SMC1b, REC8 and STAG3 protein bands were specific for testes and were not seen in cultured somatic cells, in contrast to SMC1a, which was also produced in somatic cells (see Fig. S1A in supplementary material). To confirm further the specificity of the peptide antibodies for SYCP1, SYCP2, SMC1b, REC8 and STAG3, mouse testicular cells were examined by immunofluorescence microscopy. We found that the antibodies against SYCP1, SYCP2, SMC1b, REC8 and STAG3 gave rise to a pattern that overlapped with SYCP3 in meiotic pachytene cells (data not shown). The chromosomal distribution of SMC1b, REC8 and STAG3 at MI of meiosis has been reported (Eijpe et al., 2000; Lee et al., 2003; Prieto et al., 2001; Revenkova et al., 2001). This provided us with an opportunity to verify the specificity of the anti-cohesin antibodies (supplementary material, Fig. S1B). We found that the REC8 and STAG3 proteins were localized to interchromatid domains and to the centromeres of the meiotic bivalents at MI in OA-treated cells. By contrast, SMC1b was found predominantly at the centromeres of MI male meiotic chromosomes, together with SYCP3 and SYCP2, consistent with previously published works (Eijpe et al., 2003; Prieto et al., 2001; Revenkova et al., 2001).

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Western-blot and immunofluorescence microscopy analysis of antisera. (A) Western-blot analysis of the affinity-purified guinea-pig antibodies against the SC proteins SYCP1 and SYCP2, and the cohesins REC8, STAG3 and SMC1b to whole-testis extract (T) or pachytene-cell extract (P) and somatic-cell extract (S). One strip is probed for rabbit SMC1a (Molecular Probes). Antibody against a-tubulin is used as a loading control. MW, molecular weight marker. (B) Immunolabelling of MI chromosomes derived from OA-treated wild-type spermatocytes using the guinea-pig antibodies (SYCP1, SYCP2, REC8, STAG3 and SMC1b) and the rabbit SYCP3 antisera. Each bivalent is labelled with the indicated antibody and then superimposed with DAPI and CREST antiserum to visualize chromatin and centromeres. Bars, 5 mm.

• Supplemental Figure 2 -

  Fig. S2. Colocalization of residual cohesin foci in late diplotene Sycp3^–/– oocytes with the centromeres. This is a more detailed view of data presented in Fig. 3. Oocytes are shown stained with anti-SMC1b (red), anti-REC8 (green) and anti-SMC3 (green) antibodies and antiserum against CREST (a centromere marker; green or red, to contrast with the other antibody). Boxed areas are enlarged and shown below separately for cohesin and CREST staining, with the merged image in the centre. Notice the yellow/orange colour resulting from colocalization of cohesin markers and CREST, suggesting that cohesin-complex proteins remain associated with the centromere regions.