Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Intestinal bile acids directly modulate the structure and function of C. difficile TcdB toxin

doi: 10.1073/pnas.1916965117

Figure Lengend Snippet: Cell surface binding assay. TcdB (2 nM) and either 1,000 µM TCDCA or dehydro-CA were preincubated together for 30 min on ice in serum-free media before adding to HCT116 cells. After incubating for 60 min on ice, cells were harvested and lysed. (A) Clarified material was analyzed by Western blot by probing with anti-TcdB antibody (R&D Systems, AF6246) and anti-tubulin antibody (Sigma, T6074) as a loading control. (B) Cell-associated TcdB bands were measured by densitometry using a ChemiDoc MP Imaging System (Bio-Rad). The TcdB-binding compound TCDCA, but not dehydro-CA, prevented surface binding of TcdB to cells. Bars represent SEM of three biological replicates.

Article Snippet: Clarified material was analyzed by Western blot by probing with anti-TcdB antibody (R&D Systems, AF6246) first, followed by anti-tubulin antibody (Sigma, T6074) as a loading control.

Techniques: Binding Assay, Western Blot, Control, Imaging

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Intestinal bile acids directly modulate the structure and function of C. difficile TcdB toxin

doi: 10.1073/pnas.1916965117

Figure Lengend Snippet: Identification of nonsteroidal bile acid mimetics. (A) Results from high-throughput DSF screening of 2,400 drugs from the Microsource Spectrum collection. A statistical cutoff of ΔT = 3 °C inhibition of increase in stabilization was based on identification of molecules that were greater than 3 SDs above the mean of the data. Green dots represent hits that were bile acids or bile acid-like molecules. (B) Titration of ethaverine and parent compound papaverine against TcdB by DSF. Ethaverine dose dependently binds and thermally stabilizes TcdB with greater potency than papaverine. Bars represent SEM of four experiments. (C) Titration of ethaverine against full-length and CROP-less TcdB by DSF. Ethaverine dose dependently binds and thermally stabilizes full-length TcdB but not CROP-truncated TcdB1–2,283. (D) Cell surface binding assay. TcdB (2 nM) and either 100 µM of positive control methyl cholate, 50 µM ethaverine, or 50 µM papaverine were preincubated together for 30 min on ice in serum-free media before adding to HCT116 cells. After incubating for 60 min on ice, cells were harvested and lysed. Clarified material was analyzed by Western blot by probing with anti-TcdB antibody (R&D Systems, AF6246) and anti-tubulin antibody as a loading control. Cell-associated TcdB bands were measured by densitometry using a ChemiDoc MP Imaging System (Bio-Rad). The TcdB-binding compound ethaverine and to a lesser extent papaverine, prevented surface binding of TcdB to cells. Bars represent SEM of four biological replicates. (E) Normalized transepithelial resistance measurements in human Caco-2 cells, 3 to 6 h posttreatment. Ethaverine preserved significantly increased resistance across Caco-2 monolayer cells compared to mock control values (n = 3 biological replicates). Bars represent SEM of mean. ***P < 0.0006.

Article Snippet: Clarified material was analyzed by Western blot by probing with anti-TcdB antibody (R&D Systems, AF6246) first, followed by anti-tubulin antibody (Sigma, T6074) as a loading control.

Techniques: High Throughput Screening Assay, Inhibition, Titration, Binding Assay, Positive Control, Western Blot, Control, Imaging