4116 Nucleic Acids Research, 2011, Vol. 39, No. 10 Figure 4. Validation of the identiﬁcation of hnRNP G and inter-species conservation of hnRNP G interaction with the WEc RNA. 1D-NWA of the binding of the 32P-labelled RNA WEc probe with nuclear proteins extracts from different species, followed by immunodetection using the canine anti-hnRNP G serum. (A) hnRNPG homologues in ZZ and ZW GVs of P. waltl. The 42 kDa labelled band (box) detected by NWA in ZW GVs corresponded exactly to the one detected by the anti-hnRNP G serum. Note that a band corresponding to hnRNP G was immunodetected in ZZ GVs, although no signal was visible in the corresponding blot after NWA. (B) hnRNP G from nuclear proteins extracts of human HeLa cells and GVs of the amphibians X. topicalis (X.t), N. viridescens (N.v) A. mexicanum (A.m) and P. waltl (P.w, ZW karyotype). Stars and circles indicate the bound hnRNP G. Figure 5. Interaction of the P. waltl hnRNP G isoforms with the WEc RNA. (A) 2D-NWA of the 32P-labelled RNA WEc probe with nuclear proteins from ZZ, ZW and WW GVs, followed by immunodetection using the anti-hnRNP G serum. Note that the homologues of hnRNPG formed a train of basic spots ranging from pI 9 to 10 with two major spots at pI 10. The most basic spot corresponded to the one detected by NWA in ZW and WW GVs. (B) An example of isoelectrofocusing of the protein extracts of the ZW GVs showing three spots of hnRNP G at pI 10 (arrowheads) in the silver stained gel (S). Note that only one spot was visible in the northwestern blot (N). bind the WEc RNA probe in the WW GVs, we investigated the phosphorylation state of these proteins, which may inﬂuence their RNA afﬁnity. Pro-Q diamond phospho- protein stain was used on 2D gels for a sensitive and direct in-gel detection of phosphoproteins irrespective of the phosphoamino acid (‘Materials and Methods’ section). The subsequent staining of the same 2D-gels using the total-protein SYPRO Ruby stain provided a measure of the phosphorylation level relative to the total amount of the protein. As seen in Figure 6, the two major spots corresponding to hnRNP G appeared as phosphorylated in ZZ and WW GVs. Nevertheless, the