Developmental Cell 17 Supplemental Data The E3 Ligase TTC3 Facilitates Ubiquitination and Degradation of Phosphorylated Akt

The purified recombinant products of TTC3 used for in vitro ubiquitin assays (Fig. 2A and 2B) were shown by Coomassie staining of the SDS-PAGE. Although the predicted molecular weight of the recombinant TTC3 (Amino Acid from 1540 to 2025 of human TTC3) was 55KD, the resulted recombinant proteins were migrated at approximately 70KD on SDS-PAGE because of possibly altered posttranslational modification. Method: Recombinant TTC3 was generated by pFastBac HTC baculoviral system (Invitrogen) essentially described previously (Laine et al., 2000). The subfragment of human TTC3 (Amino Acid from 1540 to 2025 of human TTC3) was amplified by PCR and subcloned into pFastBac HTC (Invitrogen). The resulted plasmid was co-transfected with Baculo-gold DNA into Sf-9 cells using Baculogold kit (PharMingen). SF-9 cells were harvested after 60 hours, lysed with the following lysis buffer [50mM Tris-HCl buffer (pH 7.5), 1 % Nonidet P-40, 10 % glycerol, 137mM NaCl, 50mM β-glycerophophate, 4µg/ml AEBSF, 4µg/ml aprotinin, 4µg/ml pepstatin A, and 4 µg/ml leupeptin], and spun at 15,000 x g for 30min. The His x 6-tagged TTC3 was purified using a Ni2+-chelating affinity column after extensive washing using LiCl2 washing buffer (lysis buffer plus 20mM imidazole and 0.5M LiCl) and eluted with elution buffer (lysis buffer plus 0.5M imidazole). The purity of the final purified recombinant TTC3 products was verified by Coomassie staining of the SDS-PAGE. Figure S2. TTC3-Akt interaction is important for efficient Akt ubiquitination (A) Diagrams of TTC3 using in the study were shown. We generated ∆AB-TTC3 (consisting of AA 1~491 and 1539~2025 of TTC3) or N-terminal TTC3 (consisting of AA 1~819 of the N-terminal TTC3), both of which lack the putative Akt-binding domain, to compare the efficiencies of ubiquitination of Akt in the cellular context. (B) Compared to wild type TTC3 transfected cells, significantly low levels of Akt ubiquitination were achieved by either ∆AB-TTC3 o