a3 vector  (ATCC)

Journal: Nucleic Acids Research

Article Title: Nuclear RNA export factor 7 is localized in processing bodies and neuronal RNA granules through interactions with shuttling hnRNPs

doi: 10.1093/nar/gkm556

Figure Lengend Snippet: NXF7 binds a series of shuttling hnRNPs. ( A ) GST (lanes 1 to 3), GST-Tap (aa 96–371; lanes 4 to 6), GST-NXF7 (full length; lanes 7 to 9), and GST-NXF7 (aa 99–374; lanes 10 to 12), each pre-adsorbed to glutathione Sepharose beads, were incubated for 2 h at 4°C with either buffer (lanes 1, 4, 7, 11), hnRNP A3 (lanes 2, 5, 8, 11) or hnRNP A2/B1 (lanes 3, 6, 9, 12). After washing extensively, bound proteins were eluted by boiling in SDS sample buffer and were then loaded on SDS-12% polyacrylamide gels. In lanes 13 and 14, ∼5% of input was loaded. Proteins were visualized by staining the gel with Coomassie brilliant blue. Migration positions of molecular weight markers are indicated on the left in kilo Daltons and those of hnRNP A3 and hnRNP A2/B1 are on the right side of the panel, indicated by arrowheads. Asterisks indicate the positions of GST and GST fusion proteins. ( B ) Same as in A, but proteins were detected by Western blot using the indicated antibodies. ( C ) Same as in A, but 6xHis-tagged KSRP was subjected to binding reactions. Proteins were detected by Western blot using anti-pentaHis antibody.

Article Snippet: Baculovirus expression vectors for mouse hnRNP A2/B1 and hnRNP A3 were constructed by inserting the corresponding cDNAs, which also had been isolated by PCR, along with GST ORF, isolated from the pGEX6P3 vector, into the pFASTBac1 vector (Invitrogen).

Techniques: Incubation, Staining, Migration, Molecular Weight, Western Blot, Binding Assay

Journal: Nucleic Acids Research

Article Title: Nuclear RNA export factor 7 is localized in processing bodies and neuronal RNA granules through interactions with shuttling hnRNPs

doi: 10.1093/nar/gkm556

Figure Lengend Snippet: NXF7 is localized in polyribosomes containing fractions. ( A ) Upper panel : Cytoplasmic lysate of a mouse L929 cell line stably expressing FLAG-tagged NXF7 was prepared using Mg ++ -containing buffer and was fractionated over a linear 20–50% sucrose gradient, which also contained Mg ++ . The ribosome profile (OD 254 nm trace) is depicted. Arrows indicate the positions of 40, 60 and 80 S ribosomal subunits in the gradient, while a bracket illustrates positions of polyribosomes. Total RNA and protein were isolated from each fraction. Distribution of tRNAs and rRNAs was examined by denaturing agarose gel electrophoresis followed by ethidium bromide staining of the gel. FLAG-tagged NXF7 fusion protein was detected by Western blot with an anti-FLAG antibody. Lower panel : Cytoplasmic lysate of a mouse L929 cell line stably expressing FLAG-tagged NXF7 was prepared using an EDTA-containing buffer. Fractionation was performed as above, but the gradient contained 50 mM EDTA. The positions of small and large ribosome subunits are shown by arrows in the OD 254 nm trace. Distribution of RNAs and FLAG-tagged NXF7 was examined as above. ( B ) Upper panel : Cytoplasmic lysate of a mouse L929 cell line stably expressing FLAG-tagged NXF7 was prepared and fractionated under Mg ++ -containing condition as in A . Lower left panel : Cytoplasmic lysate was prepared after the cells were treated with 1 mM puromycin for 5 h. Lower right panel : Cytoplasmic lysate was prepared in the presence of RNase A (100 μg/ml). In both experiments, fractionation was performed under Mg ++ -containing condition. The ribosome profiles (OD 254 nm trace) are depicted. The positions of 40, 60 and 80 S ribosomal subunits and polyribosomes in the gradients are indicated in each panel. FLAG-tagged NXF7 and hnRNP A3 in the gradients were detected by Western blot using anti-FLAG and anti-hnRNP A3 antibodies, respectively. A non-specific band that migrates slower than NXF7-FLAG is indicated by asterisks.

Article Snippet: Baculovirus expression vectors for mouse hnRNP A2/B1 and hnRNP A3 were constructed by inserting the corresponding cDNAs, which also had been isolated by PCR, along with GST ORF, isolated from the pGEX6P3 vector, into the pFASTBac1 vector (Invitrogen).

Techniques: Stable Transfection, Expressing, Isolation, Agarose Gel Electrophoresis, Staining, Western Blot, Fractionation

Journal: Nucleic Acids Research

Article Title: Nuclear RNA export factor 7 is localized in processing bodies and neuronal RNA granules through interactions with shuttling hnRNPs

doi: 10.1093/nar/gkm556

Figure Lengend Snippet: NXF7 is co-localized with hnRNP A3 in P-bodies. ( A ) HeLa cells expressing NXF7-GFP (left panels) or GFP-Dcp1a were fixed and subjected to immunofluorescence using rabbit anti-hnRNP A3 antibody. The lower left and right panels are magnified images of the areas indicated by white boxes. Localization was observed by confocal microscopy. ( B ) HeLa cells expressing NXF7-GFP and mRFP-Dcp1a were fixed and subjected to immunofluorescence as in A. Visualization of localization of hnRNP A3 was done using Alexa647-labeled anti-rabbit IgG. The lowest panel shows a magnified view of the area indicated by a white box in the merged image. ( C ) CEB-derived cells cultured on glass bottom dishes were fixed and immunostained with anti-Dcp1a and anti-NXF7 antibodies followed by Alexa568-labeled anti-rabbit and Alexa488-labeled anti-rat secondary antibodies. The cells were observed by confocal microscopy. The lower right panel shows a magnified view of the area indicated by the white box in the merged image. ( D ) Same as in C, but the cells were immunostained with anti-hnRNP A3 and anti-NXF7 antibodies. The cells were observed by confocal microscopy. The lower right panel shows a magnified view of the area indicated by the white box in the merged image. ( E ) HeLa cells expressing NXF7-GFP were cultured in the presence of 0.5 mM arsenite for 1 h. The cells were fixed and immunostained with anti-Dcp1a (upper panels) and anti-hnRNP A3 (lower panels) antibodies followed by Alexa568-labeled anti-rabbit IgG. Localization of each protein was detected by confocal microscopy.

Article Snippet: Baculovirus expression vectors for mouse hnRNP A2/B1 and hnRNP A3 were constructed by inserting the corresponding cDNAs, which also had been isolated by PCR, along with GST ORF, isolated from the pGEX6P3 vector, into the pFASTBac1 vector (Invitrogen).

Techniques: Expressing, Immunofluorescence, Confocal Microscopy, Labeling, Derivative Assay, Cell Culture

Journal: Nucleic Acids Research

Article Title: Nuclear RNA export factor 7 is localized in processing bodies and neuronal RNA granules through interactions with shuttling hnRNPs

doi: 10.1093/nar/gkm556

Figure Lengend Snippet: Co-localization of NXF7 and hnRNP A3 in distal sites of neurites. ( A ) Neuro2a cells expressing NXF7-GFP were fixed and subjected to immunofluorescence using anti-hnRNP A3 antibody. Localization was observed by confocal microscopy. Insets are magnified view of the areas indicated by the white boxes. ( B ) Same as in A, but immunofluorescence was performed using anti-Dcp1a antibody. Inset is a magnified view of the area indicated by the white box. ( C ) Neuro2a cells expressing a CFP-fusion protein containing the N+LRR domain (aa 1–374) of NXF7 was subjected to immunofluorescence using anti-hnRNP A3 antibody as in A. ( D ) Neuro2a cells expressing a CFP-fusion protein containing the M+C domain (aa 375–620) of NXF7 was subjected to immunofluorescence using anti-hnRNP A3 antibody as in A. ( E ) Neuro2a cells expressing a CFP-fusion protein containing the minimal hnRNP A3-binding domain (aa 91–374 of NXF7) was subjected to immunofluorescence using anti-hnRNP A3 antibody as in A.

Article Snippet: Baculovirus expression vectors for mouse hnRNP A2/B1 and hnRNP A3 were constructed by inserting the corresponding cDNAs, which also had been isolated by PCR, along with GST ORF, isolated from the pGEX6P3 vector, into the pFASTBac1 vector (Invitrogen).

Techniques: Expressing, Immunofluorescence, Confocal Microscopy, Binding Assay