Fig. 3. Tce1 interacts with the outer membrane receptors BtuB and OmpF.

a Identification of BtuB and OmpF as the binding partners of Tce1. Total cell lysates of Yptb YPIII were incubated with GST·Bind beads coated with GST-Tce1 or GST. After removing unbound proteins through extensive washing, the retained proteins were resolved through SDS–PAGE followed by silver staining. Protein bands specifically retained by GST-Tce1 were identified through mass spectrometry. b Interactions between Tce1 and BtuB, OmpF or TolB detected using bacterial two-hybrid assay. Interactions were assessed using MacConkey maltose plates (upper) and the β-galactosidase assay (lower). Error bars indicate ±SD (n = 3 biological replicates), with two-sided, unpaired Student’s t-test. P < 0.001 was considered as significant differences. c–e Direct binding between Tce1 and BtuB, OmpF or TolB detected using an in vitro GST pull-down assay. His₆-BtuB (c), His₆-OmpF (d), or His₆-TolB (e) was incubated with GST-Tce1, GST, or an irrelevant recombinant protein GST-CheY, and the protein complexes captured on glutathione beads were detected using western blotting. All blots were repeated for at least three times independently with similar results.