Figure 4. ADAD2 interacts with RNA-binding proteins implicated in piRNA biogenesis.

(A) Co-IP/MS analysis of ADAD2-interacting proteins. Co-IP was performed using agarose resin crosslinked with the polyclonal antibody against aa 1 – 93 of mouse ADAD2. ADAD1, ADAD2, and RNF17 are detected with high specificity in wildtype testis lysate. (B) Representative RNA-binding proteins enriched in the ADAD2-associated proteome. Blue indicates the proteins potentially involved in both piRNA biogenesis and spermatogenesis, whereas green highlights the ones implicated in spermatogenesis. (C – E) Co-IP followed by Western blot analyses. Co-IP experiments were performed using magnetic beads conjugated with the antibodies against ADAD2 (pAb), MAEL, or MILI. The immunoprecipitated proteins were then analyzed by Western blot using antibodies against ADAD1, ADAD2 (mAb#19–8), MAEL, MILI, MIWI, RNF17, and YTHDC2. Red arrowheads indicate the protein bands specifically detected in the wildtype IP samples. (F) Immunohistochemistry analyses of ADAD2 and RNF17 in wildtype and Adad2^–/– testis cryosections. ADAD2 was immunodetected using mAb#19–8 and cell nuclei were visualized by Hoechst 33342.