Figure 9. Rap1b negatively regulates... | Rockefeller University Press

Figure 9.

$Figure 9. Rap1b negatively regulates PI3K/Akt signaling via CD11b outside-in signaling. (A) Immunoblot analysis of CD11b and Rap1b in DRM fractions or WCL of Rap1b−/− or CD11b−/− neutrophils with respective WT controls. (B) Immunoblot analysis of p-Akt in WT and CD11b−/− neutrophils stimulated on uncoated plates. (C) p-Akt immunoblots analysis of WT and Rap1b−/− neutrophils that were stimulated with fMLP and plated on fibrinogen-coated slides or on slides coated with CD11b Ab. (D) Analysis of cells with more than one protrusion using F-actin staining of WT and Rap1b−/− neutrophils that were stimulated with fMLP and plated on fibrinogen-coated slides or on slides coated with anti-CD11b. (E) Immunoblot analysis of p-Akt from WT and Rap1b−/− neutrophils incubated with or without a monoclonal blocking anti-CD11b antibody after stimulation with fMLP and on fibrinogen-coated plates. Blots are representative of 3 independent experiments and densitometric analyses are cumulative of 3 experiments (A–C and E). Mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant using unpaired Student’s t test. (F) Matrix degradation analysis in CD11b−/− neutrophils or WT and Rap1b−/− neutrophils treated with blocking anti-CD11b antibody. Mean ± SD, n = 3 independent experiments (D and F). *, P < 0.05; **, P < 0.01; NS, not significant using unpaired Student’s t test. (G) Relative recruitment of CSFE and SNARF-1 labeled and adoptively transferred WT and CD11b −/− neutrophils in BAL of LPS challenged mice. Mean ± SD; n = 3 independent experiments. ***, P < 0.001 (unpaired Student’s t test).$

Rap1b negatively regulates PI3K/Akt signaling via CD11b outside-in signaling. (A) Immunoblot analysis of CD11b and Rap1b in DRM fractions or WCL of Rap1b^−/− or CD11b^−/− neutrophils with respective WT controls. (B) Immunoblot analysis of p-Akt in WT and CD11b^−/− neutrophils stimulated on uncoated plates. (C) p-Akt immunoblots analysis of WT and Rap1b^−/− neutrophils that were stimulated with fMLP and plated on fibrinogen-coated slides or on slides coated with CD11b Ab. (D) Analysis of cells with more than one protrusion using F-actin staining of WT and Rap1b^−/− neutrophils that were stimulated with fMLP and plated on fibrinogen-coated slides or on slides coated with anti-CD11b. (E) Immunoblot analysis of p-Akt from WT and Rap1b^−/− neutrophils incubated with or without a monoclonal blocking anti-CD11b antibody after stimulation with fMLP and on fibrinogen-coated plates. Blots are representative of 3 independent experiments and densitometric analyses are cumulative of 3 experiments (A–C and E). Mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant using unpaired Student’s t test. (F) Matrix degradation analysis in CD11b^−/− neutrophils or WT and Rap1b^−/− neutrophils treated with blocking anti-CD11b antibody. Mean ± SD, n = 3 independent experiments (D and F). *, P < 0.05; **, P < 0.01; NS, not significant using unpaired Student’s t test. (G) Relative recruitment of CSFE and SNARF-1 labeled and adoptively transferred WT and CD11b ^−/− neutrophils in BAL of LPS challenged mice. Mean ± SD; n = 3 independent experiments. ***, P < 0.001 (unpaired Student’s t test).