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Characterization of PML and TRIM27 Interactions with Mdm2 Through SUMOylation Analysis

This supplemental figure encompasses a detailed exploration of protein interactions between PML, TRIM27, and Mdm2, emphasizing the role of SUMOylation in these processes. Utilizing various experimental techniques, including co-immunoprecipitation and Western blot analysis, we elucidate the binding dynamics of His-SUMO1/2 and GST-tagged proteins, demonstrating their implications in cellular pathways. The data highlights the distinctive roles of TRIM proteins, their interactions, and the significance of Mdm2 regulation in the context of SUMO modifications.

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Characterization of PML and TRIM27 Interactions with Mdm2 Through SUMOylation Analysis

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  1. Supplemental Fig.1 a b V PML HA-PML - + + + + His-SUMO1 + + - + - + ++ + ++ His-SUMO2 + + + + + GST-SUMO1 - - - - + + + - + Flag-c-Jun Mdm2- SUMO2 Ni beads WCL Mdm2 c-Jun-His -SUMO1 75 Ni beads 1 2 3 4 50 c 37 His-SUMO1 c-Jun IB c-Jun- SUMO1 HA-PML - + - + 75 100 50 c-Jun- SUMO1 75 c-Jun WCL 37 PML- SUMO1 50 100 c-Jun 75 PML IB 37 GFP 1 2 3 4 1 2 3 4 5

  2. Supplemental Fig.2 a PML 150 150 100 PML 75 100 PML 50 75 actin 37 * 25 50 15 coomassie blue staining Western blot b TRIM27 Mdm2 - 100 - 75 100 Mdm2 75 Trim27 * - 50 c GST-TRIM27 GST-PML GST GST-TRIM27 GST-PML GST 1 2 3

  3. Supplemental Fig. 3 a Trim22S Trim22L Trim11 Trim22L V Trim27 V Trim39 His-SUMO1 + + + - + + + - + - + + + + HA-Mdm2 + - + + - + + + + + - + + - 150 Mdm2- SUMO1 Ni beads 100 100 Mdm2 WCL Actin GFP b Trim27 V Trim24 Trim25 V Trim18 Trim1 V + + + ++ + ++ + ++ + ++ + ++ + ++ 150 Ni beads Mdm2- SUMO1 100 100 Mdm2 WCL Actin GFP c UbcH5a - + + + TRIM27 + + + - 82 p53-Ub 64 p53 p53 1 2 3 4

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