Xenbase Image ID: 133552

Fig. 3. (A) Gel filtration of the supernatant of ultracentrifuged eggs with Sephacryl S-300HR column with HB. Two milliliters of the supernatant was applied onto the column (diameter 1.6 cm, height 55 cm) and 3 mL fractions were collected. Arrowheads show the fractions in which thyroglobuline (T; M.W. approximately 670 kDa) and catalase (C; M.W. approximately 250 kDa) were eluted, respectively. (B) Western blot analysis of gel-filtrated fractions using anti-Xtr (upper panel) and anti-FRGY2 (lower panel) rabbit antisera. The numbers and SM above the upper panel represent the fraction numbers and start material (the supernatant; 5 lg), respectively. The elute (fractions 16–19 as indicated with a bidirectional arrow in A) was pooled. (C) Western blot analysis of immunoprecipitates from pooled fractions with normal mouse IgG (NMI) (lane d) and anti-Xtr mAb (lane e). Detection of Xtr and FRGY2 was carried out by using anti-Xtr (upper panel) and anti-FRGY2 (lower panel) rabbit antisera, respectively. Xtr and FRGY2 in supernatant of centrifuged eggs (10 lg protein each) (at 15 000 g in lane a and 150 000 g in lane b) and the pooled fractions (1 lg protein) (lane c) were also detected. Note that FRGY2 was coimmunoprecipitated with Xtr. (D) Reverse transcription–polymerase chain reaction (RT–PCR) of NMI- or anti-Xtr mAb-coimmunoprecipitated mRNAs from the pooled fractions 16–19 from gel filtration using oligo dT primer for RT and gene-specific primers (RCC1, XRHAMM, XL-INCENP, XMad2, XDead end, and SP4) for PCR. W indicates total RNA (1 lg) from fertilized eggs (30 min after insemination). Spermatogenic cell-specific SP4 mRNA was added to the immunoprecipitates with NMI (NMI) and anti-Xtr mAb (a Xtr) for comparing the recovery of RNA during purification process and efficiency of RT–PCR between the immunoprecipitates. Image published in: Golam Mostafa M et al. (2009) Copyright © 2009. Image reproduced with permission of the Publisher, John Wiley & Sons. Larger Image Printer Friendly View

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