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