DEV017293 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Specificity of newly raised antibodies against Rbsn-5, Rab5, Rab11 and Rab7. (A-D) Immunostaining for Rbsn-5 in oocytes of wild-type (A,C) and rbsn-5⁻ GLC (B,D) using rabbit (A,B) or rat (C,D) polyclonal anti-Rbsn-5 antisera. Immunoreactivities were not detected in rbsn-5⁻ GLCs. (E,F) Immunostaining for Rab5 in oocytes of wild-type (E) and rab5⁻ GLC (F). No immunoreactivity was detected in rab5⁻ oocytes. DNA was counterstained with DAPI (cyan). Scale bar: 20 µm. (G) Specificity of Rab5, Rab11 and Rab7 antibodies on immunoblots. The ovarian lysates (100 µg) and purified GST-Rab5, Rab11 and Rab7 (20 ng) were subjected to immunoblot analysis with anti-Rab5, Rab11 and Rab7 antibodies.

• Supplemental Figure S2 -

  Fig. S2. Short Osk alone fails to recruit endosomal proteins and stimulate endocytosis. (A-F) Rab5 (A,B), Rab11 (C,D) and Rab7 (E,F) localization at the oocyte anterior when both Osk isoforms (A,C,E) or short Osk alone (B,D,F) were expressed. Rab5, Rab11 and Rab7 all accumulated in the anterior of oocytes expressing the unmutated osk-bcd 3′ UTR transgene that produces both long and short Osk, but not the osk(M1L)-bcd 3′ UTR transgene that produces short Osk alone (arrowheads). (G,H) Incorporation of FM4-64 in stage 9 oocytes with the unmutated osk-bcd 3′ UTR transgene (G) or the osk(M1L)-bcd 3′ UTR transgene (H). Preferential dye incorporation was never observed at the oocyte anterior in the presence of short Osk alone (arrowheads). (I,J) Kin-βgal localization with the unmutated osk-bcd 3′ UTR transgene (I) or the osk(M1L)-bcd 3′ UTR transgene (J). No ectopic Kin-βgal signal was detected at the anterior pole of the oocyte when osk-bcd 3′ UTR transgenes were expressed (arrowheads). DNA was counterstained with DAPI (cyan). Scale bars: 20 µm.