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

FIG 3.

FIG 3

Acyl-mimicking mutations at K122 of SOD1 do not affect canonical SOD1 ROS-scavenging activity. (A) HEK 293 cells were cotransfected with Flag-tagged WT SOD1 and HA-tagged WT, K122R, K122E, or K122Q SOD1 expression plasmids. Flag-WT SOD1 was immunoprecipitated (IP) with Flag-agarose resin and was subjected to SDS-PAGE before being immunoblotted for HA-tagged binding partners. (B) HEK 293 cells were transfected with a Flag-tagged WT, K122R, K122E, K122Q, or G93A SOD1 expression plasmid. Flag-tagged SOD1 was immunoprecipitated with Flag-agarose resin and was subjected to SDS-PAGE before being immunoblotted for the CCS binding partner. (C) HEK 293 cells were transfected with Flag-tagged WT, G85R, K122R, K122E, or K122Q SOD1. SOD1 was immunoprecipitated with Flag-agarose resin and was then competitively eluted with Flag peptide. The eluted SOD1 activity was then measured colorimetrically. The experiment was carried out with three biological replicates, each with three technical replicates. Asterisks indicate significant differences (**, P < 0.01; ***, P < 0.001); error bars represent SEM. (D) Samples were prepared as for the assay for which results are shown in panel C. The eluted SOD1 was resolved through native PAGE, and SOD1 activity was assayed within the gel. White bands indicate active SOD1.