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. 2020 Jun 18;11:3101. doi: 10.1038/s41467-020-16885-3

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Immunoblot of protein extracts from testes of adult mice; total lysate, cytoplasmic and nuclear fractions and immunoprecipitates with guinea pig anti-PRR19 (IP Gp-PRR19) or non-specific (IP Gp–IgG) antibodies are shown. Upper panel: immunoblot by Gp-PRR19 antibodies. Arrowhead marks presumed PRR19 band. Asterisks mark unspecific protein bands, which varied with the age of analysed mice and antibody batch (see Figs. 2c, d, 7h). Molecular weight marker positions are indicated. Nuclear histone H3 (middle panel) and cytoplasmic GAPDH (bottom panel) controlled for fractionation. b Quantification of axis-associated PRR19 foci detected by Gp-PRR19 antibody in wild-type spermatocytes in early (epa), mid (mpa), late pachytene (lpa) and diplotene (di). n = numbers of analysed cells from three animals; medians (bars) are 0 (epa), 24 (mpa), 22.5 (lpa) and 0 (di). c, d, f, h, i Immunofluorescent staining of nuclear surface-spread (c, d) wild-type spermatocytes or (f, h, i) spermatocytes of indicated genotypes from adult mice. Bars, 10 µm; in d lower panel, 5 µm. Histone H1t (c, i, insets, H1t) marks prophase stages after early pachytene. d Boxed autosome of a mid/late pachytene spermatocyte is enlarged in the bottom panel, where the PRR19 signal is shifted to the right by two pixels for better visualisation of PRR19-MLH1 co-localisation. e Quantification of co-localisation between PRR19 and MLH1 foci on chromosome axes in mid/late pachytene wild-type spermatocytes. n = numbers of analysed cells from three animals, medians (bars) are 100% (blue data set) and 96.3% (red data set). g Quantification of axis-associated PRR19 foci in pachytene or pachytene-like spermatocytes from wild types and indicated mutants. j Quantification of axis-associated PRR19 foci in mid (mpa) and late pachytene (lpa) spermatocytes of wild-type and Mlh1^Lisk/Lisk mice. g, j n = numbers of analysed cells from at least two animals; medians (bars) are (g) 24 in wild-type, 0 in Spo11^−/−, Sycp1^−/−, Mlh3^−/−, Rnf212^−/−, Hei10^mei4/mei4 and Cntd1^Q/Q, 1 in Cntd1^−/−, (j, mpa) 22 and 20, (j, lpa) 21 and 14 in wild-type and Mlh1^Lisk/Lisk, respectively. Mann–Whitney U test, **** indicates (g) P < 2.2E-16, (j, mpa) P = 5.59E-05 and (j, lpa) P = 2.43E-13. Source data are provided as a Source Data file.