Research Paper Volume 13, Issue 3 pp 3909—3925

Figure 3. USP18 imposes post-translational deubiquitination on FTO. (A, B) Determining USP18, FTO mRNA (A) and protein (B) expression upon USP18 depletion. USP18 was knocked down by transfecting USP18 siRNA-248 (si-USP-1) and USP18 siRNA-581 (si-USP-2). NC, negative control transfected with scramble shRNA. *** p < 0.001, student’s t-test. n.s., non-significant. (C) Co-immunoprecipitation determining the direct interaction between FTO and USP18. NC, negative control; OE, overexpression. (D) Co-immunoprecipitation of FTO protein and ubiquitin in the presence or absence of USP18. USP18 was knocked down by transfecting USP18 siRNA-248. (E) Western blot determining FTO protein stability in the presence or absence of USP18. USP18 was knocked down by transfecting USP18 siRNA-248. (F) Western blot showing that blockage of proteasomal degradation with MG132 stabilized FTO protein upon depletion of USP18. USP18 was knocked down by transfecting USP18 siRNA-248.