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. 2010 Sep 2;107(38):16512–16517. doi: 10.1073/pnas.1004181107

Fig. 4.

Fig. 4.

MS identification of SUMO1 attachment sites and detection of SUMO1-Ub conjugates. (A) List of proteins identified to have SUMO1 or Ub attachment sites based on MS/MS detection of the respective footprints. The target name, site of SUMO or Ub attachment, seedling treatment conditions where the footprint was detected, and whether or not the SUMO footprint is within the canonical ΨKXE attachment site (3) are listed. (B) Immunoblot detection of SUMO1 conjugates modified with Ub. WT and His-H89R-SUM1 sum1-1 sum2-1 and seedlings were collected before (0) and 30 min after a 30 min heat stress and enriched for SUMO conjugates by the 3-step affinity method shown in Fig. 2. Cr, crude seedling extracts from heat-stressed plants before purification. (C) Immunoblot detection of Ub conjugates modified with SUMO1. WT and transgenic seedlings expressing His-Ub were collected before (0) and 30 min after a 30 min heat stress and enriched for Ub conjugates by a single Ni-NTA affinity step. Start represents the crude extracts before Ni-NTA purification. Samples in (B) and (C) were subjected to SDS-PAGE and immunoblot analysis with anti-SUMO1 (Left) or anti-Ub antibodies (Right). ▸, free SUMO1. ⊳, free Ub, His-Ub and poly-Ub chains. *, large subunit of RUBISCO.