(a) Purified Arabidopsis His-AtSAE1b, His-AtSAE2, His6-AtSCE1, GST-AtSIZ1, His6-AtSUMO1 and GST-NIA1 (or NIA2)-Myc were separated by 11% SDS–PAGE. (b) Sumoylation of GST-NIA1 (or NIA2)-Myc was assayed in the presence or absence of His6-AtSAE1b, His6-AtSAE2, His6-AtSCE1, His6-AtSUMO1 and GST-AtSIZ1. After the reaction, sumoylated NIA1 or NIA2 was detected by immunoblotting with an anti-Myc antibody. AtSIZ1-mediated E3 sumoylation of NIA1 or NIA2 was examined in Arabidopsis. Leaves of WT (c) or siz1-2 plants (d) were infiltrated with 35S-Myc-AtSIZ1, 35S-HA3-AtSUMO1 and 35S-FLAG-NIA1 (or NIA2), as indicated. After incubation for 3 days, FLAG-NIA1 (or NIA2) was detected by immunoblotting with an anti-FLAG antibody. Sumoylated NIA1 or NIA2 was detected by immunoblotting with an anti-FLAG antibody after IP with an anti-HA antibody. (e) To confirm the size of the non-sumoylated or sumoylated form of NIA1, total proteins were extracted from the leaves of WT plants infiltrated without or with 35S-FLAG-NIA1 construct. Total proteins were also extracted from the leaves of WT plants infiltrated without or with 35S-Myc-AtSIZ1, 35S-HA3-AtSUMO1 and 35S-FLAG-NIA1, and were immunoprecipitated with an anti-HA antibody. Total proteins or immunoprecipitated proteins were separated by 11% SDS–PAGE and detected by immunoblotting using an anti-FLAG antibody. IgG, immunoglobulin G.