Figure 7. Molecular characterization of... | Rockefeller University Press

Figure 7.

$Figure 7. Molecular characterization of the function of BopN in MAPK pathways. (A) Immunoblot analysis of MAPK activities in the lysates of DC2.4 cells. DC2.4 cells were stimulated with 10 µg/ml LPS or infected with the indicated Bordetella strains for the specified time periods. The lysates were analyzed by Western blotting with antibodies against phospho-p38 (top), phospho-ERK1/2 (third from top), and phospho-JNK (fifth from top). The membranes were stripped and reblotted with antibodies against p38 (second panel), ERK1/2 (fourth from top), and JNK (bottom). (B) Immunofluorescence microscopy of DC2.4 cells stimulated with LPS for 30 min or infected for 30 min with the indicated strains. DC2.4 cells were fixed and stained with anti–phospho-ERK1/2 (green), anti-ERK1/2 (red), and DAPI (blue). Data are representative of three independent experiments. Bar, 20 µm. (C) 10 µM of MEK1/2 inhibitor U0126, 50 µM of p38 kinase inhibitor SB203580, and vehicle control (DMSO) were added to DC2.4 culture medium for 1 h before infection (m.o.i. = 100) with the indicated strains. Total RNA was prepared from the treated cells, and the amounts of IL-10 mRNA produced were assessed by qRT-PCR. (D) DC2.4 cells were infected with the indicated strains (m.o.i. = 100) for the specified time periods, and the amounts of phospho-MEK1/2 and phospho-MKK3/MKK6 were analyzed using immunoblotting. Three independent experiments were performed and a representative immunoblot is shown. (E) DC2.4 cells were stimulated with 10 µg/ml LPS or infected with the indicated strains for 30 min, and nuclear fractions were prepared and analyzed using a Multiplex transcription factor profiling kit. Differences in transcription factor activation were determined by Bio-Plex. The values in C and E are means ± SE from three independent experiments. *, P < 0.05.$

Molecular characterization of the function of BopN in MAPK pathways. (A) Immunoblot analysis of MAPK activities in the lysates of DC2.4 cells. DC2.4 cells were stimulated with 10 µg/ml LPS or infected with the indicated Bordetella strains for the specified time periods. The lysates were analyzed by Western blotting with antibodies against phospho-p38 (top), phospho-ERK1/2 (third from top), and phospho-JNK (fifth from top). The membranes were stripped and reblotted with antibodies against p38 (second panel), ERK1/2 (fourth from top), and JNK (bottom). (B) Immunofluorescence microscopy of DC2.4 cells stimulated with LPS for 30 min or infected for 30 min with the indicated strains. DC2.4 cells were fixed and stained with anti–phospho-ERK1/2 (green), anti-ERK1/2 (red), and DAPI (blue). Data are representative of three independent experiments. Bar, 20 µm. (C) 10 µM of MEK1/2 inhibitor U0126, 50 µM of p38 kinase inhibitor SB203580, and vehicle control (DMSO) were added to DC2.4 culture medium for 1 h before infection (m.o.i. = 100) with the indicated strains. Total RNA was prepared from the treated cells, and the amounts of IL-10 mRNA produced were assessed by qRT-PCR. (D) DC2.4 cells were infected with the indicated strains (m.o.i. = 100) for the specified time periods, and the amounts of phospho-MEK1/2 and phospho-MKK3/MKK6 were analyzed using immunoblotting. Three independent experiments were performed and a representative immunoblot is shown. (E) DC2.4 cells were stimulated with 10 µg/ml LPS or infected with the indicated strains for 30 min, and nuclear fractions were prepared and analyzed using a Multiplex transcription factor profiling kit. Differences in transcription factor activation were determined by Bio-Plex. The values in C and E are means ± SE from three independent experiments. *, P < 0.05.