Journal:
Article Title: A Compartmentalized Phosphorylation/Dephosphorylation System That Regulates U snRNA Export from the Nucleus ▿ †
doi: 10.1128/MCB.01189-07
Figure Lengend Snippet: PHAX is phosphorylated by CK2 in vivo. (A) HeLa cells were transfected with the plasmid expressing either Flag-tagged WT mPHAX (Flag-mPHAX WT) or the ΔST2 mutant (ΔST2). After 24 h, the lysate of the transfected cells either was left untreated (−) or was treated with CIAP (+), and Western blot analysis was performed with anti-Flag antibody. Phosphorylated (P) and underphosphorylated (under-P) forms of Flag-mPHAX are indicated. Double bands are indicated by two dots. A degradation product of PHAX is indicated by the asterisk. (B) HeLa cells were treated with siRNAs against CK2α and CK2α′ (lanes 2 to 5) or the control siRNA (Control; lane 1) for 72 h and subsequently were transfected with the plasmid expressing Flag-mPHAX WT. For lanes 3 to 5, either the vector (lane 5) or the plasmids expressing Myc-tagged CK2α (lane 3) or CK2α′ (lane 4) that contained silent mutations conferring resistance to the siRNAs also were transfected with the Flag-mPHAX plasmid. After 24 h, the total cell lysate was prepared from the transfected cells and was analyzed by Western blotting using antibodies denoted on the left side of the figure. The identities of detected bands are indicated on the left side (for endogenous CK2α and CK2α′) or on the right side (for all other proteins). (C) HeLa cells either left untransfected (top row), transfected with Flag-mPHAX WT (middle row), or transfected with Flag-mPHAX ΔNLS+NES (bottom row) were treated with various concentrations of staurosporine (Sigma) for 3 h. The treated cells were analyzed by Western blotting with anti-PHAX antibody (top row) or anti-Flag antibody (middle and bottom rows). P and under-P represent phosphorylated and underphosphorylated forms of PHAX, respectively. The percentage of the underphosphorylated form is shown below each panel. The asterisk indicates a degradation product of PHAX. (D) HeLa cells transfected with Flag-mPHAX WT (images a and b) or Flag-mPHAX ΔNLS+NES mutant (images c and d) were analyzed by indirect immunofluorescence using an anti-Flag antibody (images a and c). Images b and d show DNA staining by Hoechst 33342 of images a and c, respectively.
Article Snippet: The following antibodies were used for Western blot analysis: anti-CK2α/α′ (BD Biosciences), anti-CK2β 6D5 (Calbiochem), anti-PP2A-A 4G7 (Upstate Biotechnology Inc.), anti-PP2A-B 2G9 (Upstate Biotechnology Inc.), PP2A-C 7A6 (Upstate Biotechnology Inc.), anti-nucleolin 4E2 (MBL), anti-hnRNP-C1/C2 4F4 (Sigma), anti-Flag M2 (Sigma), anti-Myc MC045 (Nacalai Tesque), anti-GAPDH 6C5 (Ambion), and anti-PHAX polyclonal antibody ( 29 ).
Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot, Immunofluorescence, Staining
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![Subcellular localization of CK2 and PP2A subunits. (A) Western blotting analysis of nuclear (N) and cytoplasmic (C) fractions from HeLa and NIH 3T3 cells and Xenopus oocytes with antibodies against various subunits of CK2 and PP2A. Antibodies against two nuclear proteins (nucleolin and hnRNP C1/C2) and a cytoplasmic protein (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were employed for checking the quality of the fractionations. The N and C fractions from equivalent cell numbers are compared for the left three columns, whereas a 10 times greater number of N fractions [N(×10)] were loaded for the right-most column. (B) Confocal laser-scanning microscopy of HeLa cells stained with anti-CK2α (a) and anti-PP2A-B (c) antibodies. Images b and d show DNA staining by Hoechst 33342 of images a and c, respectively.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3276/pmc02223276/pmc02223276__zmb0010872210007.jpg)