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. 2017 Sep 26;37(20):e00354-17. doi: 10.1128/MCB.00354-17

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FIG 5 — Development of an antibody specific to acylated K122 of SOD1. SIRT5 inhibition results in succinylation of endogenous SOD1. (A) Flag-tagged WT or K122R SOD1 was overexpressed in HEK 293 cells with or without SIRT5 knocked down by siRNA. Flag-tagged SOD1 was immunoprecipitated with Flag-agarose resin, subjected to SDS-PAGE, and immunoblotted for succinyl-K122 (Su-K122) and total (Flag) SOD1. (B) Endogenous SOD1 was immunoprecipitated from HEK 293 cells with or without SIRT5 knocked down via siRNA. The immunoprecipitated SOD1 was then subjected to SDS-PAGE and immunoblotted with the Su-K122 antibody. The experiment was carried out in triplicate. The asterisk indicates a significant difference (*, P < 0.05); error bars represent SEM. (C) Flag-tagged WT or K122R SOD1 was overexpressed in HEK 293 cells with or without SIRT5 knocked down by siRNA. Oxygen rates were measured with an Oroboros O2K respirometer. The experiment was carried out in triplicate. Asterisks indicate significant differences (**, P < 0.01; ***, P < 0.001).