C. difficile Toxin B/TcdB Antibody

Detection ofC. difficileToxin B/TcdB by Western Blot. Western blot shows recombinantC. difficileToxin B/TcdB (Catalog # 6246-GT). PVDF membrane was probed with 1 µg/mL of Sheep Anti-C. difficileToxin B/TcdB Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6246) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for Toxin B/TcdB at approximately 75 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of C. difficile Toxin B/TcdB by Western Blot Effective TcdB cell interactions correlate with endocytosis. (A, B) Representative confocal maximum-intensity projections of CHO-K1 cells stained with 250 nM Alexa Fluor 647-labeled TcdB1 B2′B3 (red) (A) or Alexa Fluor 647-labeled TcdB2 B2′B3 (red) (B). Ten micromolar calcein AM (green) was used to counterstain the cytoplasm of live cells with intact membranes, and 0.5 μg/ml Hoechst 33258 (blue) was used to visualize the nucleus. (C) Quantification of the images in panels A and B, expressed as corrected total cell fluorescence. (D) Representative immunoblotting of full-length TcdB1 and TcdB2 associated with CHO-K1 cells in the presence or absence the dynamin inhibitor dynasore (80 μM). Incubations were carried out for 30 min at 37°C. (E) Quantification of data presented in panel D, expressed as relative band density. (F) Representative immunoblotting of full-length TcdB1 and TcdB2 associated with CHO-K1 cells when incubations were carried out for 30 min at a temperature that permits (37°C) or inhibits (ice) endocytosis. (G) Quantification of data presented in panel F, expressed as relative band density. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28776043), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of C. difficile Toxin B/TcdB by Western Blot Mechanism-of-action of niclosamide inhibition of TcdB intoxication. c In vitro auto-processing assay. Compound or DMSO was added to 100 nM TcdB, and pre-incubated for 60 min. To initiate auto-processing, InsP6 was added (100 μM) and incubated @ 37 °C for 20 min before stopping with Laemlii sample buffer. Niclosamide at concentrations up to 10 μM did not affect auto-processing (n = 2). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30531960), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of C. difficile Toxin B/TcdB by Western Blot Impact of PepB2 on TcdB enzymatic activity, thermal stability, and cell association. (G) Immunoblot analysis of TcdB associating with cells for 30 min. CHO-K1 cells were exposed at 37°C to 4 nM TcdB in the presence and absence of 50 µM peptide. The cells were then washed, and total cell lysates were examined by immunoblotting. In this figure, all bar graphs represent the mean densitometry value ± standard deviation, and asterisks indicate significant change. *, P < 0.01; **, P < 0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28512094), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of C. difficile Toxin B/TcdB by Western Blot Mechanism-of-action of niclosamide inhibition of TcdB intoxication. a Western blot image for intracellular Rac1 glucosylation (n = 3). IMR-90 cells were treated with niclosamide (doses indicated) for 15 min, followed by treatment with 0.5 pM TcdB. Cells were harvested as described in Methods. Mab102, which recognizes un-glucosylated Rac1 in cells, shows a dose-dependent re-appearance with increasing concentration of niclosamide, relative to total Rac1 levels (n = 2). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30531960), licensed under a CC-BY license. Not internally tested by R&D Systems.