ALKBH5 depletion has a minor effect on m6A levels in poly(A) RNA. (A) LC-MS/MS measurements of m6A, m6Am, and inosine in poly(A) RNAs isolated from WT and ALKBH5 KO cells. (B) Immunoblot analysis of protein expression in 293T, HeLa, U87, and U2OS cells transfected with control (scrambled) or ALKBH5 targeting siRNAs (ALKBH5 KD). The levels of proteins were detected with specific antibodies listed in Materials and Methods. (C) LC-MS/MS measurements of m6A, m6Am, and inosine in poly(A) RNAs isolated from WT and ALKBH5 KD in 293T, HeLa, U87, and U2OS cells. 293T (n = 9), HeLa, U87, and U2OS (n = 3). (D) Immunoblot analysis of protein expression in 293T cells overexpressing ALKBH5 (ALKBH5 OE) or GFP control. The levels of proteins were detected with specific antibodies listed in Materials and Methods. (E) LC-MS/MS measurements of m6A, m6Am, and inosine in poly(A) RNAs isolated from control (GFP) and ALKBH5 overexpressing cells. Data are mean ± SD, one-way ANOVA, multiple comparison with the Tukey test (for ALKBH5 KD). Data are mean ± SD, unpaired two-tailed t-test (for ALKBH5 KO and ALKBH5 OE). (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. The levels of nucleoside modifications were normalized to the total molar amount of canonical nucleosides N (N = C + U + G + A). Source data are available online for this figure.
