Fig. 4
From: Modification of messenger RNA by 2′-O-methylation regulates gene expression in vivo
Nm modification of mRNA codon inhibits translation. a, b 293T WT and U32A + U51 KO cells transfected with myc-tagged Pxdn and labeled with AHA for 4 h. Plots show mean ± SEM. a AHA-labeled protein detected by biotin click-labeling. Plot shows quantification, normalized to total protein (*p = 0.0083). b PXDN protein captured by anti-myc IP, then click-labeled with biotin and detected by immunoblotting (arrow). Plot shows quantification of biotinylated PXDN normalized to relative mRNA expression and total biotinylated protein from Fig. 4a (*p = 0.0485). c Mutation of Pxdn A3150 to C3150 (red) creates a base-pair mismatch at the putative methylation site. d 293T WT cells were transfected with myc-tagged Pxdn WT or C3150 mutant. Cells were AHA labeled for 4 h, then nascently translated PXDN protein was captured, biotinylated, and quantified as in b. n = 7/group, across three independent experiments. Plot shows mean ± SEM, *p = 0.0119 vs. WT by unpaired t-test. Representative immunoblot shown, including relative Pxdn mRNA expression levels (RQ)
