Fig. 5.

HSPA2 is an interaction partner of MOV10L1. (A) Identification of MOV10L1-interacting partners by pull-down experiment with recombinant MOV10L1. Forty-eight hours before harvesting, COS cells were Mock-transfected (left lane) or transfected with a plasmid expressing Flag-tagged MOV10L1 (center and right lanes). Lysates were then incubated with anti-Flag antibody and Sepharose A (left and right lanes) or only with Sepharose A (center lane). Flag-tagged MOV10L1 bound to the Sepharose was then incubated with lysates from P20 mouse testis. Proteins bound to MOV10L1 and Sepharose were separated by SDS-PAGE. After silver staining of the gel, protein bands that were visible only in the right lane, but not in the two control lanes, were cut out and analyzed by MALDI-TOF mass spectrometry. HSPA2, heat shock 70-kDa protein 2; IgG, Ig; hc, heavy chain; lc, light chain. (B) COS1 cells were cotransfected with expression plasmids for Flag-tagged MOV10L1 and Myc-tagged HSPA2. Lysates were then used for coimmunoprecipitation with anti-Flag antibodies followed by Western blot analysis with anti-Myc antibodies. Input lanes represent 1% of lysates used for coimmunoprecipitation. Reciprocal coimmunoprecipitation worked similarily. (C) Model for the role of MOV10L1 during spermatogenesis. MOV10L1 interacts with MILI and MIWI, which is necessary for piRNA-induced repression of retrotransposons in pachytene spermatocytes. MOV10L1 interacts further with HSPA2.