pcag ires gfp vector  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9

doi: 10.1074/jbc.RA118.002890

Figure Lengend Snippet: Rpl29 methylation shows no effect on global protein synthesis. A, Western blotting showing the absence of Rpl29 in Rpl29−/− NIH 3T3 clones generated by CRISPR-Cas9. B, exogenous Rpl29 or the K5R mutant (untagged) was stably expressed in Rpl29−/− NIH 3T3 cells, and the cell lysates were immunoblotted with Rpl29 antibody and D8T9P, respectively. C, SUnSET assay showing global protein synthesis in the indicated cell lines. As a control for protein synthesis inhibition, NIH 3T3 cells were treated with cycloheximide (CHX, 100 μg/ml) for 12 h before the addition of puromycin. D, [35S]methionine labeling assay showing global protein synthesis in the indicated cell lines. Shown are the mean ± S.D. from three independent experiments. The significance of differences was determined with one-way analysis of variance (ANOVA). *, p < 0.05; ns, not significant. E, polysome profiling showing similar fractions of mRNPs and polysomes in all cell lines.

Article Snippet: J1 mESCs or NIH 3T3 cells were transiently co-transfected with the pCAG-Cas9-IRES-GFP vector and two synthesized gBlocks (Integrated DNA Technologies) containing the U6 promoter and Rpl29 targeting sequences in intron 1 and exon 4, respectively ( Fig. S1 A ).

Techniques: Methylation, Western Blot, Clone Assay, Generated, CRISPR, Mutagenesis, Stable Transfection, Inhibition, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Neurogenin 2 Mediates Amyloid-β Precursor Protein-stimulated Neurogenesis *

doi: 10.1074/jbc.M114.581918

Figure Lengend Snippet: Transfection of NSPCs with a Ngn2 cDNA induces neuronal differentiation. Cells were cultured for 5 days in differentiation medium. A, NSPCs derived from APPKO mice were transfected either with empty vector pCAG-IRES-GFP (Control) or with pCAG-Ngn2-IRES-GFP (Ngn2). The figure shows 3 independent representative immunofluorescence images for each group. Transfected cells (green) are marked with asterisks. Cells were stained for β-III-tubulin (red). No double-stained GFP+ β-III-tubulin+ cells were observed in empty vector incubation, indicating that cells transfected with the empty vector are not neurons. In cells transfected with the Ngn2 plasmid, there was co-staining of GFP and β-III-tubulin, indicating that each cell transfected with Ngn2 was neuronally differentiated. B, quantification of the results in panel A shows the % of β-III-tubulin+ GFP+ cells to total GFP+ cells. C, representative immunofluorescence images of NSPCs derived from WT mice transfected with siRNA-control-Cy3 vector or siRNA-Ngn2-Cy3 vector (Ngn2). The figure shows siRNA (red), β-III-tubulin (green), and DAPI (blue) staining. Cells transfected with the control plasmid (61) showed co-staining of siRNA and β-III-tubulin. Cells transfected with the Ngn2 siRNA (Ngn2), marked with arrows, were not β-III-tubulin+ (asterisk). D, quantification of the results in C showing the % β-III-tubulin+ siRNA+ cells to total siRNA+ cells. F, western blotting analysis of Ngn2 levels. The figure shows a representative western blot (E) and quantification (F). Decreased expression of Ngn2 was observed in NSPCs after siRNA interference. Cells were cultured for 5 days in differentiation medium. β-Actin was used as a control for protein loading. Scale bars = 50 μm. Data are expressed as the means ± S.E. (** = p < 0.001; *** = p < 0.0001 as determined by Student's t test). Cont, control; Ngn2, neurogenin 2.

Article Snippet: Plasmid pCAG-Ngn2-IRES-GFP for the expression of Ngn2 was a kind gift of François Guillemot (MRC-National Institute for Medical Research, London, UK), and the plasmids pCAG-IRES-GFP control vector and pCAG-IRES-APP695 were from Addgene (Cambridge, MA).

Techniques: Transfection, Cell Culture, Derivative Assay, Plasmid Preparation, Immunofluorescence, Staining, Incubation, Western Blot, Expressing