4114 Nucleic Acids Research, 2011, Vol. 39, No. 10 human hnRNP G/RBMX (accession number P38159) and the mouse hnRNP G (accession number O35479) proteins (Figure 3, Supplementary Tables S1 and S2). These results suggested the presence in the ZZ, ZW and WW GVs extracts of PwhnRNP G protein isoforms with closely similar molecular masses and isoelectric points but showing differential binding activity for the WEc RNA probe according to the karyotype of the oocyte. Further conﬁrmation of the PwhnRNP G identity of the 42 kDa polypeptides by RNA binding and western blot assays In order to conﬁrm the identiﬁcation of the 42 kDa poly- peptides as the homologues of the human hnRNP G protein, WEc RNA-binding assays followed by immuno- detection assays with canine autoantibodies sera, which had been shown to react monospeciﬁcally with the human hnRNP G protein (26), were performed sequen- tially on blots of proteins from P. waltl GVs separated by mono-dimensional electrophoresis. No signal was immunodetected when the serum was pre-absorbed with the human hnRNP G (Supplementary Figure S1). In contrast, the canine anti-hnRNP G autoantibodies serum recognized one major labelled band in P. waltl GVs (Figure 4A). This band showed a perfect superimpos- ition with the labelled 42 kDa band present in the corres- ponding northwestern blots with the ZW, WW GVs and Hela extracts (Figure 4A and B). In the case of the ZZ GVs an immunoreactive band was also detected at the same position but no radioactive signal was observed in the corresponding NWA. This result conﬁrmed the presence of PwhnRNP G proteins in the 42 kDa band, which differentially bound the WEc RNA in the three P. waltl karyotypes. We further investigated whether hnRNP G homologues from other vertebrate species could also interact with the WEc RNA by carrying out similar assays on protein extracts from GVs of the amphibians X. tropicalis, Ambystoma mexicanum and Notophthalmus viridescens. As shown in Figure 4B, in all three cases one band of nearly 42 kDa was labelled by the WEc RNA probe and showed a perfect superimposition with the immuno- detected band. Altogether, these results indicated that the afﬁnity of the hnRNP G protein for the WEc RNA is conserved among vertebrate species. Correlation of the WEc RNA-interacting isoforms of the PwhnRNP G protein with the sex genotype of the P. waltl oocyte Northwestern and immunodetection assays performed in parallel on the same 2D western blot further conﬁrmed the identiﬁcation of the 42 kDa/pI 10 P. waltl polypeptides as hnRNPG homologues (Figure 5A). The position of the immunodetected spots corresponded to the polypeptides showed by mass spectrometry to be the homologues of the human hnRNPG protein. Furthermore, the western blot analysis revealed the existence in the oocytes of the three karyotypes, of a basic train of isoforms, ranging from pI 9 to 10 with closely similar molecular weights and present in different amounts. Three major spots were visible, one at pI 9 and the other two at pI 10, which could be distinguished from one another by a slight variation in molecular weight and pI. The superim- position of the autoradiogram with the western blot showed that none of the hnRNP G homologues bound the WEc RNA probe in the ZZ GVs because no radio- active hybridization signals were detected in the corres- ponding area of the autoradiogram. In contrast, the most basic hnRNP G polypeptide in the ZW and WW GVs showed an exact match with the radioactive signal detected in the corresponding autoradiogram reﬂecting its binding to the WEc RNA probe. This binding was con- ﬁrmed to be sequence-speciﬁc by the lack of hybridization signal with the PP6, BP7, BP7 RNA probes (data not shown). Occasionally and depending on the degree of resolution of the IEF in the basic pI region, three major closely migrating spots were found in the pI 10 area of the silver stained 2D-gels with ZW GVs instead of the two more frequently observed. The two more basic very close spots were of the same molecular mass while the third more acidic spot was of a lower molecular mass. The superimposition of the northwestern blot autoradio- gram with the matching immunodetection blot showed that the WEc RNA probe bound the most basic polypep- tide only (Figure 5B). We attempted to achieve a higher IEF resolution to determine whether the pI and molecular mass of the hnRNP G isoforms in the ZZ GVs were slightly different from those in the WW GVs. To this end we used the 2D DIGE ﬂuorescence labelling method to reduce gel-to-gel variations. Samples of ZZ and WW GVs proteins were pre-labelled with different ﬂuorescent dyes and co-migrated in the same gel. The superimposition of the ﬂuorescent images did not reveal any difference for these isoforms between the two karyotypes (data not shown). Taken together, these results demonstrated that the polypeptides of 42 kDa and pI 10 observed in the ZZ, ZW and WW of P. waltl GVs did correspond to three PwhnRNP G isoforms in the ZW GV and two isoforms in the ZZ and WW GVs. Only the most basic isoform bound to the WEc RNA probe in a sequence speciﬁc manner in the ZW and WW GVs. The phosphorylation of PwhnRNP G is not required for binding the WEc RNA The theorical isoelectric point of the human hnRNPG predicted from its primary protein sequence is 10.06. As shown in Figure 5A, the 2D migration proﬁle of its immunodetected homologues in P. waltl oocytes extracts showed a train of spots with nearly identical molecular weights and isoelectric points ranging between pI 9 and pI 10. These pI shifts could result from post-translation modiﬁcations (PTMs) such as phosphorylation, acetyl- ation or methylation, which are known to play a signiﬁ- cant role in the regulation of gene expression by modifying protein–protein and/or nucleic acids–proteins inter- actions. Phosphorylation is one of the most common PTM that can be detected by a direct in-gel method. Because the hnRNP G isoforms of the ZZ GVs did not bind the WEc RNA probe and differed in their ability to