Figure 3. Detection of RME-8 by Western blot analysis (A) and RME-8 localization in ovary by indirect immunofluorescence staining (B-E). A protein band (asterisk) corresponding to the size of the predicted RME-8 (260 kDa) was detected in wild type (A). This band was present in rme-8
) worms reared at the permissive temperature, but it was greatly reduced (<) in rme-8
) worms that were shifted to the restrictive temperature for 48 h. In addition to the wild-type band, a larger band (No.) corresponding the size of the predicted RME-8::GFP protein (290 kDa) was observed in the RME-8::GFP strain. Equal amounts of total worm proteins were loaded on each lane. (B-E) Confocal images of wild-type (B and C) and rme-8
) (D and E) ovaries stained with anti-RME-8 antibodies. Images were obtained with the same exposure parameters focusing on the middle focal plane at low magnification (B and D) or on the cortical focal plane at high magnification (C and E). Wild-type RME-8 is localized to the oocyte cortex (B) on punctate structures (C). Mutant RME-8 carrying the b1023
mutation shows a diffuse cytoplasmic staining (D) and less localization to the punctate structures (E). Worms are shifted to the restrictive temperature for 24 h.