HITS-CLIP analysis of ALKBH8 RNA targets in HEK293 T-Rex Flp-In cells identifies a broader RNA binding repertoire. (A) Western blot analysis of the doxycycline-inducible expression of stably integrated 3x FLAG-ALKBH8. Tubulin was used as a loading control. (B) FLAG-ALKBH8 localizes in the cytoplasm in HEK293T. Immunofluorescence of FLAG-ALKBH8 whose expression was induced by doxycycline and visualized by anti-FLAG and Alexa594 antibodies (red). DNA was stained with DAPI (blue). The scale bar represents 50 µm. (C) ALKBH8 is present mainly in the cytoplasm. Western blot analysis of the total cell lysate (I), cytoplasmic (C), and nuclear (N) fractions of HEK293T cells. ALKBH8 was detected with a specific antibody. Tubulin was used as a cytoplasmic marker and fibrillarin (FBL) as a nuclear marker. (D) ALKBH8-RNA complexes formed by UV treatment. Cellular lysates were treated with an increasing concentration of RNaseI (marked above the gel). CTRL was a negative control of HEK293T in which FLAG-ALKB8 was not expressed. ALKBH8 was immunoprecipitated with anti-FLAG antibody. RNA was 5′-terminally labeled with γ32P-ATP, and complexes were resolved on polyacrylamide and SDS-PAGE denaturing gels, respectively. The upper panel displays the radioactive signal of the RNA detected by autoradiography, and the lower panel is a western blot analysis with anti-FLAG antibody. (E) RNA classes identified by ALKBH8 HITS-CLIP experiments. The graph shows numbers of uniquely mapped reads (x-scale).
