Nucleic Acids Research, 2011, Vol. 39, No. 10 4117 Figure 6. Relationship between PwhnRNPG phosphorylation state and RNA-binding activity. (A) Protein extracts of ZZ and WW GVs were separated in 2D-gels and stained with two sequential ﬂuorescent dyes. Pro-Q Diamond (in blue) was speciﬁc of the phosphorylated proteins and Sypro Ruby (in red) revealed all proteins. A molecular mass indicator, PeppermintStick (PM), which contained phosphorylated proteins, b-casein (BC), ovalbumin (OA) and an non-phosphorylated protein, bovine serum albumin (BA) was run in parallel with each gel to show the speciﬁcity of the staining. The box indicates the major forms (a and b) of PwhnRNP G at pI 10. A high magniﬁcation of the merge of the two ﬂuorescent stains is shown on the side of each gel. (B) Representative quantiﬁcation of the phosphorylation degree of the major forms of PwhnRNP G (a and b) based on the intensity of their ﬂuorescent staining by Pro-Q Diamond. Values were normalized relatively to the protein quantity estimated from the Sypro Ruby staining. (C) RNA-binding activity of dephosphorylated PwhnRNP G. Dephosphorylated ZZ (ZZd) and WW (WWd) or non-dephosphorylated (ZZ and WW) GV extracts were submitted to monodimensional NWA using WEc RNA probe followed by immunodetection (ID) of PwhnRNP G using the anti-hnRNP G serum. The arrow points to the PwhnRNPG protein. Note that a band corresponding to PwhnRNP G was immunodetected in ZZ GV, although no signal was visible in the corresponding blot after NWA in dephosphorylated or non-dephosphorylated extracts. degree of phosphorylation of the WW-b isoform spot, which bound the WEc RNA probe, was lower than that of the WW-a, ZZ-a and ZZ-b isoforms, which did not bind this probe. In order to study a possible functional relationship between the phosphorylation degree of PwhnRNPG and its ability to bind the WEc RNA, protein extracts from ZZ and WW GVs were treated with alkaline phosphatase. The