FIG. 3.

Confirmation of interaction between S. marcescens pirin and PDH E1. (A) In vitro GST pull-down assay followed by SDS-PAGE (panel i) and Western blot analysis (panel ii) using anti-His-Tag antibody was performed to confirm the interaction between GST-pirin_Sm and His-PDH E1. GST-pirin_Sm and His-PDH E1 were oversynthesized in E. coli before the assay. Lane 1, GST-pirin_Sm with His-PDH E1; lane 2, GST-NlpB_Sm with His-PDH E1; lane 3, GST Tag with His-PDH E1; lane 4, GST-pirin_Sm only; lane 5, GST-NlpB_Sm only; lane 6, GST Tag only; lane 7, E. coli(pBG20) spent crude extract containing His-PDH E1; arrow, His-PDH E1; lane M, protein markers (same as those in Fig. 2). (B) Oversynthesized GST-PDH E1 was used as the bait to confirm its interaction with pirin_Sm. After interaction, SDS-PAGE (panel i) and Western blot analysis using anti-pirin_Sm polyclonal antibody (panel ii) were performed. Lane 1, GST-PDH E1 with CH-1 lysate; lane 2, GST-NlpB_Sm with CH-1 lysate; lane 3, GST Tag with CH-1 lysate; lane 4, spent CH-1 cell lysate; lane 5, GST-NlpB_Sm only; lane 6, GST Tag protein only; lane 7, spent crude extract containing GST-PDH E1 from E. coli DH5α; arrow, pirin_Sm. (C) Bacterial two-hybrid assay. Colony color changed from colorless to pink-red after transformation of both pSC17 (pKT25 plasmid containing the pirin_Sm gene) and pSC18 (pUT18 plasmid containing the PDH E1 aceE_Sm gene) into E. coli DHM1, indicating specific activation of maltose catabolic genes and interaction between GST-pirin_Sm and His-PDH E1 (14). Zip25 and Zip18 were used as positive controls.