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. 2021 May 28;49(10):5779–5797. doi: 10.1093/nar/gkab415

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — ROS induces ALKBH5 phosphorylation and SUMOylation by activating ERK/JNK signaling. (A) Denaturing IP analysis indicating ROS significantly induces ALKBH5 phosphorylation and SUMOylation in HEK293T cells. (B) Western blot analysis indicating ROS dramatically activates the ERK/JNK signaling pathway in HEK293T cells. (C) Denaturing IP assay indicating inhibition of ERK dramatically blocks ROS-induced ALKBH5 phosphorylation and SUMOylation. (D) Denaturing IP analysis suggesting inhibition of JNK significantly inhibits ROS-induced ALKBH5 phosphorylation and SUMOylation. (E) IP analysis showing that ROS induces ALKBH5 phosphorylation at serine residues S87 and S325. (F) Denaturing IP analysis indicating that blocking ALKBH5 phosphorylation markedly inhibits ALKBH5 SUMOylation. Strep-tagged wild-type ALKBH5 or phosphorylation-deficient ALKBH5 was overexpressed alone or co-expressed with HA-tagged PIAS4 and Flag-tagged SUMO-1 in HEK293T cells. Denaturing IP assays were performed to determine the effect of blocking ALKBH5 phosphorylation on ALKBH5 SUMOylation. (G) Co-IP analysis showing that blocking ALKBH5 phosphorylation inhibits the interaction between ALKBH5 and SUMO E2 UBC9. Flag-tagged UBC9 was overexpressed alone or co-expressed with Strep tagged wild-type or phosphorylation-deficient ALKBH5 in HEK293T cells and Co-IP assays were performed to determine the effect of blocking ALKBH5 phosphorylation on the interaction between ALKBH5 and UBC9. (H) Dot-blot analysis indicating that blocking ALKBH5 phosphorylation dramatically increases ALKBH5 mRNA m⁶A demethylase activity. Strep tagged wild-type or phosphorylation-deficient ALKBH5 was expressed in HEK293T cells. Two days after transfection, the cells were collected and subjected to dot-blot analysis.