PMC

(A) Y2H assays showing interaction of CLDN16-WT with CLDN19-WT and CLDN19-WT with CLDN19-WT, but not of CLDN16-WT with CLDN16-WT. Shown are plates with selective medium lacking leucine and tryptophan (–LW), indicating the transforming of both bait and prey vectors; with SD-LWHA, indicating the expression of reporter genes HIS3 and ADE2; and β-galactosidase assay (A615 values) for quantification of interaction strength. (B) Mutations in CLDN19 affecting its homomeric interaction. (C) Mutations in CLDN19 affecting its heteromeric interaction with CLDN16. (D) Mutations in CLDN16 affecting its heteromeric interaction with CLDN19.

Detection of β-galactosidase activity from pTomβgal 4 expression in E. coli. Cells were harvested and extracts were prepared every 12 h and the A₆₁₅ was measured. Cultures were grown with the addition of the chromogenic substrate X-Gal (open symbols) or X-Gal and the transcriptional inducer IPTG (closed symbols) in the medium. The vector used as a positive control for E. coli β-galactosidase activity was pGEM (▪), and the vector used as a negative control and for expression was pFLAG-CTC either without (○, •) or with (⋄, ♦) the pTomβgal 4 ORF.

Elution profiles of pigmented material (A₄₀₅) containing KatY from a cell extract of Y. pestis KIM10 (substrain D28) during stepwise chromatography on columns containing DEAE-cellulose (A [the insert illustrates details of the area of KatY elution shown in the box]), calcium hydroxylapatite (B), and Sephadex G-150 (C). A₂₈₀ (○), A₄₀₅ (●), and A₆₁₅ for Blue dextran (▴) were used to determine the void volume for the column containing Sephadex G-150. See Materials and Methods for details.

The assays were carried out at 30°C in pH-MM^Suc to monitor sugar fermentation by A₆₁₅ (A, B, and C) or in MM^Pyr to monitor bacterial growth by OD₆₁₅ (D, E, and F). Bacteria were exposed to mirandamycin (A and D), compound 2 (B and E) or compound 3 (C and F) at concentrations ranging from 14 to 383 µM. Replicate assay is shown in Figure S2.