Figure 1.

A) A western blot analysis with anti-SUMO 2/3 antibody (ab3742) is shown following the preparation of a non-denaturative protein lysates (as described in section 2) with or without the addition of an isopeptidase inhibitor NEM. The absence of NEM results in a significant loss of high molecular weight (HMW) SUMO-conjugates (as shown, for example, for the two proteins indicated by arrowheads). The positions of free SUMO2 and SUMO3 and the migrating positions of MW markers are indicated.

B) Increasing the amount of anti-SUMO1 antibody (10, 30, 60 µl, 2, 6, 12 µg of antibody, respectively, (sc-5308 AC)) results in a better IP of sumoylated proteins; 1mg of protein in 600 µl of lysate was used. WCL-whole cell lysate.

C) Analysis of KAP1 sumoylation. Whole-cell lysate (WCL), negative control (Igg) and IP fractions are shown. The migrating positions of molecular weight (MW) markers are indicated. IP with anti- KAP1 antibody (ab 22553), followed by SUMO1(ab32058) and KAP1 western blot analyses identified high molecular weight SUMO-conjugates above a non-modified isoform of KAP1 (a band around 100 kDa, asterisk).

D) A cross-linkage of antibodies to the beads facilitates the elimination of the heavy and light chains in the eluate. An example of a SUMO2/3 IP analysis performed with and without anti-SUMO 2/3 antibody (ab3742) cross-linkage is shown.

E) To confirm the possible sumoylation of CDK1, an in vitro sumoylation reaction was performed with a recombinant GST- CDK1 protein, sumoylation enzymes (E1, E2), and either normal (N) SUMO or a mutant (M) SUMO incapable of forming an isopeptide bond (SUMOlink™ SUMO-1 kit from Active Motif (40120)). Western blot analysis with an anti-CDK1 antibody (Santa Cruz, sc-54) revealed the presence of a sumoylated CDK1 band above the non-modified CST-CDK1 when using the normal (N) but not mutant (M) SUMO isoform.