Dp201 01

The coding sequences of YUC8/REIN7 and truncated YUC8/REIN7 (loss of 47 amino acid residues, YUC8/REIN7m) were amplified by PCR using the primers described in S1 Table and then linked to the T-vector for sequencing. The correct YUC8/REIN7 and YUC8/REIN7m coding sequences were cloned into the expression vector pGEX-6p-1 using the BamH I and Sal I sites and fused with a GST-tag. Next, the constructed vectors were transformed into the E. coli BL21 (DE3) strain, and the transformed strains were cultured in Luria–Bertani (LB) medium and harvested after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16°C for 8 h. The fusion proteins were extracted with the lysis buffer containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 1% (vol/vol) Tween 20, and 20% (wt/vol) glycerol. The recombinant proteins were purified using a ProteinIso GST Resin according to the manufacturer’s instructions (Transgen, DP201-01). The concentration of the purified recombinant proteins was determined using the bicinchoninic acid protein assay (CWBIO, CW0014). The components of the purified proteins were analyzed by SDS-PAGE electrophoresis and Western blotting. The purified protein was immediately divided into aliquots and frozen in liquid nitrogen, and stored at -80°C for the further experiments.

E. coli Transetta (DE3) cells were transformed with pGEX-4T-1-Drp1, pGEX-4T-1-Ube1, pET-28a-Ubch10, pET-28a-Ubch5, pET-28a-Ubc4, pET-28a-Ube2s, pET-28a-Ube2k, pET-28a-Ubc7, pET-28a-Apc2 and pET-28a-Apc11, respectively. The protein expression was induced by 0.5 mM IPTG for 16 h at 16 °C. Cell lysates were prepared by repeated freezing-thawing method with liquid nitrogen and water bath of 28 °C. Lysates were incubated with GST Resin (TransGen Biotech DP201-01) or Ni-NTA Resin (TransGen Biotech DP101-01) overnight at 4 °C. GST-DRP1 and GST-UBE1 were eluted with 20 mM GSH. His-UbcH10, His-UbcH5, His-Ubc4, His-UBE2S, His-UBE2K, His-Ubc7, His-APC2 and His-APC11 were eluted with 300 mM imidazole. Eluted proteins were concentrated to 3 mg/ ml with Amicon Ultra-0.5 centrifugal filter units (Millipore). Protein aliquots were frozen in liquid nitrogen immediately and then stored at −80 °C.

For co-IP assay, 293T cells cotransfected with Flag-tagged and HA-tagged protein were lysed in immunoprecipitation buffer (50 mM Tris–HCl, pH7.4, with 250 mM NaCl, 1 mM EDTA, 50 mM NaF, and 0.5% Triton X-100, together with protease inhibitors). The Flag-tagged proteins were precipitated with anti-Flag M2 resin following the manufacturer’s instructions (Sigma, M8823). For GST pull-down assays, Escherichia coli were lysed in lysis buffer (10 mM Tris pH7.4, 150 mM NaCl, 0.25% NP-40, 10 mM EDTA, 5 mM DTT, 5 mM PMSF, and protease inhibitors) by sonication. GST-tagged proteins precipitated by affinity column chromatography were carried out using amylose resin following the manufacturer’s instructions (Transgene, #DP201-01). His-tagged proteins precipitated by affinity column chromatography were carried out using nickel column following the manufacturer’s instructions (Qiagen, 30210). Purified His-PKM2 protein was incubated with amylose resin containing GST-tagged protein for 2 h at 4 °C and eluted with 10 mM GSH in 50 mM Tris (pH = 8.0). For immunoblotting, proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Immunoblots were developed using Western Lightning Chemiluminescence Reagent Plus (Advansta) and exposed with chemiluminescence machine (36 ).