MID1 catalyzed the ubiquitination of PP2Ac.

<p><b>A.</b> Western-blot (WB) showing <i>in</i><i>vitro</i> 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>B.</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. <b>C.</b> 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. <b>D.</b> 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. <b>E.</b> Ubiquitination of PP2Ac in the presence of 10 different E2 conjugating enzymes.