Fig. 4

Ck137956 regulates ubiquitination and localization of Miwi. A Co-IP assay of interaction between HA-Ck137956 and Flag-Miwi in HEK-293 T cells. B Co-IP assay of interaction between Ck137956 and Miwi in testis with or without RnaseA/T treatment. C Domain mapping of the Ck137956-Miwi interaction by co-transfection with HA-tagged Ck137956 truncations and Flag-tagged Miwi truncations. The scheme of Ck137956 and Miwi truncations is shown in the upper panel. The grey indicates disordered region. D The levels of Miwi protein expression in day-24 Ck137956^+/- and Ck137956^−/− testis. Tubulin was used as a loading control. n = 3. Data were presented as the mean ± SEM. **p < 0.01. E mRNA expression levels of Miwi quantified by qRT-PCR in day-24 Ck137956^+/- and Ck137956^−/− testis. n = 3. Data were presented as the mean ± SEM. NS, not significant. F Overexpression of HA-tagged ubiquitin in HEK-293 T cells and analysis of the ubiquitylation of Miwi with or without Ck137956 overexpression by immunoprecipitation using anti-HA antibody. The cells were harvested after a 6-h treatment with 20 μM of MG132. G Seminiferous tubule sections were stained with Miwi (red) and DAPI (blue). Pachytene spermatocytes and round spermatids were indicated by red dotted box and solid box, respectively. Scale bar: 20 μm. H Venn diagram showing the overlap among down-regulated genes in Ck137956^−/− and Miwi^−/− RS, and target genes of Miwi