Fig. 2: ULK1 R170 is symmetrically dimethylated by PRMT5 and demethylated by KDM5C.

a–h Immunoblot were performed with the indicated antibodies. a LN229 cells with or without expression of PRMT5 shRNA were incubated with 1% oxygen for 12 h. b LN229, Huh7 or HOK cells were incubated with 1% oxygen for 12 h. Immunoprecipitations were performed using an anti-PRMT5 antibody. c Purified WT His-ULK1 or His-ULK1 R170K protein was mixed with purified WT Flag-PRMT5 or Flag-PRMT5 R368A protein for an in vitro methylation assay. d LN229 cells were pretreated with or without 200 mM IOX1 for 12 h, followed by incubation under 1% oxygen for 12 h. e LN229, Huh7 or HOK cells were cultured in normoxia condition. Immunoprecipitations were performed using an anti-ULK1 antibody. f LN229 and HOK cells with or without expression of KDM5C shRNA were incubated with 1% oxygen for 12 h. g LN229 with expression of His-ULK1 were incubated under 1% oxygen for 12 h. His-ULK1 proteins were pulldown, the precipitates were mixed with purified WT Flag-KDM5C or Flag-KDM5C H514A mutant protein for an in vitro demethylation assay in normoxia condition. h LN229 with expression of His-ULK1 were incubated under 1% oxygen for 12 h. His-ULK1 proteins were pulldown, washed and mixed with purified WT Flag-KDM5C protein for an in vitro demethylation assay in presence of indicated concentrations of oxygen. i Purified Flag-KDM5C protein was mixed with ULK1 R170me2s peptide for an in vitro demethylation assay in presence of indicated concentrations of oxygen. Data represent the mean ± SD from three independent experiments. Source data are provided as a Source data file.