observed with the WEc RNA. The other RNA probes (PP6, PB7 and BP7) did not bind to these polypeptides although they bound to other polypeptides for all three oocyte karyotypes. These data provided evidence for sequence-speciﬁc RNA-binding polypeptide(s) whose expression in the oocyte could be correlated with the presence of the W chromosome in its karyotype. Characterization of the 42 kDa polypeptide as a homologue of the human hnRNPG/RBMX protein by mass spectrometry In order to identify the 42 kDa polypeptide(s), GVs proteins from ZZ, ZW and WW oocytes were separated in duplicated bi-dimensional gels. One gel, to be used for preparative purposes, was stained to detect all proteins while the other was transferred to a nitrocellulose membrane, which was incubated with the WEc RNA probe (northwestern blot) and processed for autoradiog- raphy. Two pH ranges of immobilized gradients (IPG) Nucleic Acids Research, 2011, Vol. 39, No. 10 4113 were used for the ﬁrst dimension. A wide pH range (pH 3–10) of IPGs was used initially, but no obvious dif- ference in the pattern of protein spots labelled with the WEc probe was detected between the three types of GVs in NWA (data not shown). However, with a pH 6–11 gradient signiﬁcant differences were found in the basic pI region. As shown in Figure 2, only one labelled poly- peptide spot was detected in the 42 kDa/pI 10 region by the WEc RNA in the case of the ZW and WW GVs. This labelled spot was absent in the ZZ GVs extract contrary to other labelled spots of higher molecular mass, which were present for all three karyotypes, and are used as internal controls. Interestingly, extrapolation of the position of this 42 kDa/pI 10 spot to the matching silver-stained 2D-PAGE gels pointed to at least two closely migrating polypeptides that were present not only in the ZW and WW GVs but also in the ZZ GVs (Figure 2B, lower panels). Each of the corresponding spots was excised sep- arately from the gels and submitted to mass spectrometry analysis. They were identiﬁed as homologues of the Figure 2. Characterization of the WEc RNA-interacting 42 kDa polypeptides. (A) Six IEF-2D PAGE gels of ZZ, ZW and WW GVs proteins were prepared using 13 cm wide pH 6–11 IEF gel strips. IEF was followed by the separation in the second dimension, which was carried out simultan- eously for the six gels. For each karyotype, one gel was processed for NWA using the WEc RNA as a probe, while the other was silver stained. Note that some spots (circles and double circles) were observed in the ZZ, ZW and WW northwestern blots. The one (double circles) was identiﬁed by LC-MS-MS as the DEAD (Asp-Glu-Ala-Asp) box polypeptide 17 (Homo sapiens, ATP-dependant RNA helicase DDX17) (Supplementary Table S3). In contrast, the 42 kDa and pI 10 spots (arrows) were observed in the ZW and WW and not in the ZZ northwestern blots although the corres- ponding polypeptides were visible in the stained gel. The spots of interest (42 kDa, pI 10) were cut out from the gel and processed for mass spectrometry. (B) High magniﬁcation of the areas corresponding to the spots of interest from the northwestern blots (ZZ, ZW and WW) and the stained gels (ZZ0, ZW0 and WW0).