Journal: PLoS ONE

Article Title: SUMOylation of GTF2IRD1 Regulates Protein Partner Interactions and Ubiquitin-Mediated Degradation

doi: 10.1371/journal.pone.0049283

Figure Lengend Snippet: (A) Schematic diagram of human GTF2IRD1 protein structure and its various domains. The peptide regions used to map protein binding sites are indicated by the annotated thick lines above and the C-terminal truncations (TR1–3) are indicated below. The domains (from left to right) include a leucine zipper (LZ, purple), the repeat domains (RD1–5, gold), SUMO attachment sites (SUMO1 & 2, black), nuclear localization signal (blue), polyserine tract (green) and the conserved C-terminal domain (maroon). (B) Yeast 2-hybrid assays confirming and mapping the GTF2IRD1 interaction with PIASxβ. Double transformations were performed using pGADT7-PIASxβ plus the control empty pGBKT7 bait plasmid (CTR), pGBKT7-GTF2IRD1 full-length (FL), or the peptide regions indicated in A or the C-terminal truncation fragments (TR1–3). Positive interactions are indicated by survival on QDO plates and blue α-galactosidase staining. Survival on DDO plates was tested for all double transformants to ensure the presence of bait and prey plasmids in the yeast host (C) A similar assay testing the binding of GTF2IRD1 to UBC9 and mapping the interaction sites.

Article Snippet: The universal, normalized, mouse cDNA yeast 2-hybrid library (Cat. No. 630483, Mate & Plate, Clontech) was screened according to the manufacturer's instructions using pGBKT7-GTF2IRD1 as the bait plasmid.

Techniques: Protein Binding, Plasmid Preparation, Staining, Binding Assay

Journal: PLoS ONE

Article Title: SUMOylation of GTF2IRD1 Regulates Protein Partner Interactions and Ubiquitin-Mediated Degradation

doi: 10.1371/journal.pone.0049283

Figure Lengend Snippet: (A) Yeast 2-hybrid assays showing relative survival and α-galactosidase activation of yeast colonies on QDO and DDO plates. The yeast cells were co-transformed with pGADT7-ZMYM5 and a variety of bait plasmids including an empty pGBKT7 vector control (CTR), full-length GTF2IRD1 and the individual domains of GTF2IRD1. (B) Schematic diagram of ZMYM5 showing the position of 2 SUMO-interacting motifs (SIMs) and the MYM-type zinc finger domains (M). The black bars below indicate the extent of the ORF found in the ZMYM5 clone isolated in the original yeast 2-hybrid screen (Y2H CDS) and the truncated forms (TR99–TR486) generated to map the interaction domain. (C) Yeast 2-hybrid analysis of GTF2IRD1 interaction domain in ZMYM5 using the truncation series (TR99–TR486). (D) Immunofluorescence analysis of COS-7 cells transfected with pEGFP-ZMYM5 and pMyc-GTF2IRD1 showing extensive colocalization in the nucleus as illustrated by the overlay of GFP, anti-Myc immunofluorescence and DAPI (MERGE). (E) Western blot analysis of protein extracts from HEK293 cells co-transfected with pEGFP or pEGFP-ZMYM5 and pMyc-GTF2IRD1 plasmids. Whole cell extracts (INPUT) or proteins immunoprecipitated using anti-GFP antibody (IP) were immunoblotted (IB) with anti-Myc and anti-GFP antibodies. GFP-ZMYM5 and GFP were detected at approximately 100 kDa and 25 kDa respectively. Asterisks indicate IgG heavy and light chains.

Article Snippet: The universal, normalized, mouse cDNA yeast 2-hybrid library (Cat. No. 630483, Mate & Plate, Clontech) was screened according to the manufacturer's instructions using pGBKT7-GTF2IRD1 as the bait plasmid.

Techniques: Activation Assay, Transformation Assay, Plasmid Preparation, Isolation, Generated, Immunofluorescence, Transfection, Western Blot, Immunoprecipitation