Extended Data Fig. 5: Oxidative regulation of SlALKBH2 appears to be conserved.
From: Redox modification of m6A demethylase SlALKBH2 in tomato regulates fruit ripening
a, Protein similarity assay revealing a conserved peptide sequence at the N terminus of SlALKBH2 homologues across 15 plant species. The Cys36 and Cys39 sites of SlALKBH2 are highlighted in the conserved peptide sequence, and the type of amino acid (aa) in the homologues are shown. b, AtALKBH9B and OsALKBH9 form homodimers under oxidation by H2O2. c, The mutation of Cys37 in AtALKBH9B and Cys49 in OsALKBH9 impaired the formation of homodimers. d, Quantification of homodimer versus monomer shown in c (-DTT). For b and c, the SlALKBH2 homologues or their mutated variants were expressed in N. benthamiana leaves treated with 100 mM H2O2 for 30 min. e, The mutation of Cys37 in AtALKBH9B or Cys49 in OsALKBH9 resulted in an obvious decrease in their protein levels. f, Protein levels are quantified as shown. The SlALKBH2 homologues or their mutated variants were expressed in N. benthamiana leaves. For d and f, data are means ± s.d. (n = 3 biological replicates). Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t-test). NS, no significance. g, AtALKBH10B proteins failed to form homodimers upon H2O2 exposure. AtALKBH10B-HA was expressed in N. benthamiana leaves treated with 100 mM H2O2 for indicated times. h, Mutation of the cysteine residues in AtALKBH10B did not impact its protein levels. i, Quantification of protein levels shown in h. AtALKBH10B-HA or its mutated variants were expressed in N. benthamiana leaves for 36 h. For b, c, e, g and h, total proteins were extracted and subjected to immunoblot using an anti-HA antibody. Actin was used as a loading control. For i, data are means ± s.d. (n = 3 biological replicates). Statistical significance was assessed through one-way ANOVA analysis (P < 0.05, Tukey’s test).
