human integrin α x β 2  (R&D Systems)

Journal: mBio

Article Title: The Extracellular Domain of the β 2 Integrin β Subunit (CD18) Is Sufficient for Escherichia coli Hemolysin and Aggregatibacter actinomycetemcomitans Leukotoxin Cytotoxic Activity

doi: 10.1128/mBio.01459-19

Figure Lengend Snippet: β 2 integrin β subunit is necessary for U-937 cell sensitivity to HlyA cytotoxic activity. (A) PEG-precipitated HlyA was incubated at various concentrations with the U-937 wild-type and individual-integrin-subunit-knockout cell lines at 2 × 10 6 cells/ml for 1 h. Cells were washed, and cell viability was measured by XTT assay. The percentage of cytotoxicity was normalized to Triton X-100-treated cells at 100% and RPMI-only-treated cells at 0%. The CD 50 was calculated in GraphPad Prism, and bars represent the average and SEM from 3 biological replicates. One-way ANOVA with Bonferroni’s multiple-comparison test was performed in GraphPad Prism, with significance of each cell type compared to U-937 cells represented. (B) As in panel A, a standard XTT cytotoxicity assay was performed with multiple-integrin-subunit-knockout cell lines as indicated. Results were normalized and statistics determined as described above. (C) β 2 expression was assessed by flow cytometry on intact cells from the cell lines indicated. Bars represent the average mean fluorescent intensity (MFI) and SEM from 3 biological replicates with at least 50,000 events recorded per replicate. One-way ANOVA with Bonferroni’s multiple-comparison test was performed in GraphPad Prism, with the significance of each cell type compared to U-937 Δβ 2 cells represented. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Recombinant integrin pairs and human ICAM-1 were separated by a 4 to 20% gradient SDS-PAGE gel, transferred to nitrocellulose, and probed with HlyA at 1 μg/ml. (Left) Bound HlyA was detected with polyclonal anti-HlyA, and integrin α subunits were detected with monoclonal antibodies. (Center) Bound HlyA was detected with a pool of monoclonal anti-HlyA antibodies, and the integrin β 2 subunit was detected with a polyclonal antibody. (Right) Single-channel images of the center blot. Multiplexed near-infrared fluorescence was used to detect multiple proteins on the same blot using a Licor Odyssey imager. Blots are representative of three biological replicates.

Article Snippet: Recombinant human integrin α L β 2 (3868-AV), human integrin α M β 2 (4047-AM), human integrin α X β 2 (5755-AX), and human ICAM-1/CD54 (720-IC) were obtained from R&D Systems.

Techniques: Activity Assay, Incubation, Knock-Out, XTT Assay, Comparison, Cytotoxicity Assay, Expressing, Flow Cytometry, Recombinant, SDS Page, Bioprocessing, Fluorescence

Journal: mBio

Article Title: The Extracellular Domain of the β 2 Integrin β Subunit (CD18) Is Sufficient for Escherichia coli Hemolysin and Aggregatibacter actinomycetemcomitans Leukotoxin Cytotoxic Activity

doi: 10.1128/mBio.01459-19

Figure Lengend Snippet: β 2 integrins specifically enhance the activity of LtxA. (A, B, and D) PEG-precipitated LtxA was incubated at various concentrations with U-937 wild-type, individual- or multiple-subunit-knockout cell lines, or complemented Δβ 2 cells as indicated for each panel at 2 × 10 6 cells/ml for 3 h. Following toxin incubations, cells were washed, and cell viability was measured by XTT assay. The percentage of cytotoxicity was normalized to Triton X-100-treated cells at 100% and RPMI-only-treated cells at 0%. The CD 50 was calculated in GraphPad Prism, and bars represent the average and SEM from 3 biological replicates. The dashed line indicates the limit of detection. One-way ANOVA with Bonferroni’s multiple-comparison test was performed in GraphPad Prism, with significance of each cell type compared to U-937 cells represented. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) β 2 expression was assessed by flow cytometry on intact cells from the cell lines indicated. Bars represent the average MFI and SEM from 3 biological replicates with at least 50,000 events recorded per replicate. One-way ANOVA with Bonferroni’s multiple-comparison test was performed in GraphPad Prism, with significance of each cell type compared to U-937 cells represented. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) Recombinant integrin pairs and human ICAM-1 were separated by a 4 to 20% gradient SDS-PAGE gel, transferred to nitrocellulose, and probed with LtxA at 1 μg/ml. Bound LtxA was detected with cross-reactive monoclonal anti-HlyA antibodies. The integrin β 2 subunit was detected with a polyclonal antibody. Multiplexed near-infrared fluorescence was used to detect multiple proteins on the same blot using a Licor Odyssey imager. Blot is representative of three biological replicates.

Article Snippet: Recombinant human integrin α L β 2 (3868-AV), human integrin α M β 2 (4047-AM), human integrin α X β 2 (5755-AX), and human ICAM-1/CD54 (720-IC) were obtained from R&D Systems.

Techniques: Activity Assay, Incubation, Knock-Out, XTT Assay, Comparison, Expressing, Flow Cytometry, Recombinant, SDS Page, Fluorescence