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. 2005 May 13;102(21):7760–7765. doi: 10.1073/pnas.0500778102

Fig. 4.

Fig. 4.

In vitro and in vivo assays indicate that AtSIZ1 is a SUMO E3 ligase. (A) In planta sumoylation profiles of wild-type and siz1-2 seedlings that were grown for 10 days on 1× MS medium at 24°C (24) and then exposed to a 30-min heat shock of 40°C (40). (B) Analysis of in vitro sumoylation profiles indicates that AtSIZ1 facilitates production of ScSmt3-ScCdc3 conjugates. The indicated amount of GST-AtSIZ1 (0, 2, 4, or 10 μg) or ScSIZ2 (0 or 2 μg) was added to the reaction mixture and incubated with 10 mM ATP at 37°C for 0 (inc. –) or 90 min (inc. +) as described in ref. 23. Immunoblot analysis was performed with anti-ScSmt3 (Upper) or anti-T7 (detecting T7-ScCdc3; Lower). Filled arrowheads indicate bands corresponding to ScSmt3-ScCdc3 conjugates. Open arrowheads identify the AtSIZ1-ScSmt3 and ScSiz2-ScSmt3 conjugates. An asterisk identifies the T7-ScSIZ2-His protein. Numerals identify the migration position and molecular mass of the markers (kilodaltons).