Journal: PLoS ONE
Article Title: MID1 Catalyzes the Ubiquitination of Protein Phosphatase 2A and Mutations within Its Bbox1 Domain Disrupt Polyubiquitination of Alpha4 but Not of PP2Ac
doi: 10.1371/journal.pone.0107428
Figure Lengend Snippet: A. Western-blot (WB) showing in vitro ubiquitination of PP2Ac by MBP-tagged full-length MID1. (1) Lanes 1 and 2 show the results of ubiquitination assay for control reactions. In lane 1 ATP was omitted (no reaction) and in lane 2, only MBP was used. Lane 3 contains the results for the MPB-tagged MID1-catalyzed reaction. Two strong shifted bands corresponding to mono- and di-ubiquitinated PP2Ac and a faint smearing pattern indicative of poly-ubiquitinated PP2Ac were observed. (ii) Lane 1 shows the Biorad visible marker and lane 2 shows a control reaction in which MBP-MID1 was omitted. Lane 3 shows the ubiquitinated PP2Ac to confirm the molecular weights of the mono- and di-ubiquitinated products. B. (i) Ubiquitination of PP2Ac by MBP-tagged MID1 in the absence and presence of full-length alpha4. (ii) Ubiquitination of PP2Ac with increasing amounts of alpha4. The amounts of alpha4 were present at 1:1, 1:2 and 1:4 molar ratios with PP2Ac. C. Ubiquitination of PP2Ac by the MID1 RB1B2 protein (RING-Bbox1-Bbox2, MID1 residues 1–214). Lanes 1–6 show the results of control experiments in which a specific component of the assay was omitted, including the E1 activating enzyme (E1) (lane 1). Mono- and di-ubiquitinated PP2Ac were observed in lane 7, with all components of the reaction present. Background noise precludes detection of polyubiquitinated products. Antibody was specific for PP2Ac. D. Ubiquitination of PP2Ac by various constructs of the three N-terminal domains. These domains were expressed and purified similarly, and used at roughly the same concentration for the assays. Lane 1 uses the Bbox1-Bbox2 construct that includes the amino acids from the linker region between the RING and Bbox1 domains (LB1B2, residues 71–115). Lane 2 uses a RING domain (residues 1–92) that also includes a large position of the linker region. The RB1 and RB1B2 constructs consist of residues 1–164 and 1–214 respectively. E. Ubiquitination of PP2Ac in the presence of 10 different E2 conjugating enzymes.
Article Snippet: Human Ub-activating enzyme E1, human Ub-conjugating enzymes (UbcH5b), UbcH (E2) enzyme, and biotinylated ubiquitin were purchased from BostonBiochem (Bio-Techne Inc., Minneapolis, MN).
Techniques: Western Blot, In Vitro, Ubiquitin Assay, Marker, Construct, Purification, Concentration Assay
R&D Systems is a verified supplier 
R&D Systems manufactures this product 
