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. 2014 Jun 2;289(29):20273–20282. doi: 10.1074/jbc.M114.579821

FIGURE 4.

FIGURE 4.

RNAi knockdown of JGT leads to a decrease of base J and changes in hmU and fU. A, relative expression of JGT mRNA levels using quantitative RT-PCR in JGT RNAi cells with our without tetracycline 36 h after induction. B, anti-base J dot blot. DNA isolated from the indicated cell lines was serially diluted by 2-fold and spotted onto a nitrocellulose membrane to measure base J levels as described under “Experimental Procedures.” The membrane was then hybridized with a radioactive tubulin probe to control for DNA loading. 90-13, RNAi cell line untransfected with the JGT-pZJM construct; −Tet, uninduced JGT RNAi cells; +Tet, induced with tetracycline for the indicated days. C, genomic DNA isolated from JGT RNAi cells uninduced (−Tet) and at the indicated days following induction with tetracycline was analyzed by LC-MS/MS as described under “Experimental Procedures.” We detected a slight increase in levels of fdU in the tetracycline-induced samples compared with the uninduced samples (day 1.5, p = 0.5751; day 3.5, p = 0.1872).