Constructed Gal4AD and Gal4BD vectors were transformed into the yeast two-hybrid strain PJ69-4a (James et al., 1996 (link)) in pairs and allowed to grow at 30°C for 2–3 days. Transformants were selected on SD-Leu-Trp plates. Protein interaction was determined on synthetic complete medium lacking Trp, Leu and His, supplemented with 3-amino-1,2,4-triazole (3-AT, Sigma-Aldrich), or on plates lacking Trp, Leu, and Ade. SD-Leu-Trp plates were used as a control.
Full-length BdUEV1 ORFs were cloned in plasmid pGEX-6p and BdUBC13s were cloned in plasmid pET30a as previously described (Guo et al., 2016 (link)). The His6 and GST fusion constructs were transformed into E. coli strain BL21 (DE3) and the recombinant proteins were purified with Ni Sepharose and glutathione (Amersham Pharmacia), respectively. For the pull-down assay, crude cell extracts were loaded on glutathione SepharoseTM 4B beads and then 10 μg of purified His6-BdUbc13 was later added. After incubation and washing, the GST beads were boiled with SDS-PAGE loading buffer for 10 min prior to Western blotting.
Full-length BdUEV1 ORFs were cloned in plasmid pGEX-6p and BdUBC13s were cloned in plasmid pET30a as previously described (Guo et al., 2016 (link)). The His6 and GST fusion constructs were transformed into E. coli strain BL21 (DE3) and the recombinant proteins were purified with Ni Sepharose and glutathione (Amersham Pharmacia), respectively. For the pull-down assay, crude cell extracts were loaded on glutathione SepharoseTM 4B beads and then 10 μg of purified His6-BdUbc13 was later added. After incubation and washing, the GST beads were boiled with SDS-PAGE loading buffer for 10 min prior to Western blotting.
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