FIGURE 3.

(A) Xiwi1 and Xili are predominantly present in small complexes. Lysate prepared from (upper panel) stage I/II and (lower panel) stage III/IV oocytes was gel-filtered as described previously, on separate columns (Minshall et al. 2007). Alternate fractions were resolved by 15% SDS-PAGE and analyzed by Western blot with the indicated antibodies. For stage I/II fractions, Xiwi1 was detected using the anti-Miwi antibody (Deng and Lin 2002). For stage III/IV fractions, the Western blot was probed with the anti-Xiwi1 and anti-Xili antibodies described above. The molecular weight standards used were chymotrypsin A (25 kDa), ovalbumin (43 kDa), aldolase (158 kDa), catalase (232 kDa), and ferritin (440 kDa). (B) Xiwi1 and Xili do not interact with the cap-binding complex. m⁷GTP-Sepharose chromatography was performed using stage III/IV oocyte lysate. Following binding, the beads were washed and then eluted with GTP- and m⁷GpppG-containing buffer and finally with SDS buffer. Input represents ∼0.25% of the input lysate; the flow-through and wash fractions represent 1% of each fraction; the GTP and m⁷G fractions represent 12% of each fraction; and SDS represents 50% of the SDS fraction.