Figure 4. Defective TLR3- and TLR4-induced activation of IRF3 in Lyst-mutant Bg-J cells. (A) WT and Bg-J BMDMs were stimulated with 10 µg/ml poly(I:C) for the indicated times. Whole cellular lysates were immunoblotted (IB) for phosphorylated IRF3 (p-IRF3), p-TBK1, or p-Erk1/2. Reprobing with anti-IRF3, TBK1, and Erk1/2, respectively, served as the loading control. The arrowhead indicates p-IRF3. (B) Whole cellular lysates from WT and Bg-J BMDMs were immunoblotted with antibodies against TRAM, TRIF, TRAF3, and Rab7. Immunoblotting with actin-specific antibodies served as a loading control. (C) WT and Bg-J BMDMs were stimulated with 100 ng/ml LPS for the indicated times. TLR4 endocytosis was measured by flow cytometric assessment of the loss of TLR4 cell-surface expression. Data are mean ± SD from duplicate cultures. (D–F) BMDMs from WT and Bg-J mice were stimulated with 100 ng/ml LPS for the indicated times. (D) Whole cellular lysates were immunoblotted for p-Erk1/2, p-p38, p-JNK, p-IκBα, and IκBα. Blots were reprobed for total Erk2, p38, and actin to verify equal protein loading. (E) Immunoblot analysis as in D using antibodies specific for p-IRF3. p-IRF3 is indicated by an arrowhead. Reprobing with anti-IRF3 served as the loading control. (F) Nuclear extracts were prepared and analyzed by immunoblotting for p-IRF3 and total IRF3. Blots were reprobed for GAPDH (cytoplasmic marker) and lamin A/C (nuclear marker). Whole cellular lysate from WT BMDMs served as a control. (G and H) WT and Bg-J BMDMs were treated with 10 µg/ml poly(I:C) or 100 ng/ml LPS or left unstimulated (unstim.). After 4 h of stimulation, cell-surface expression of CD86 was determined by flow cytometry. (G) Representative flow cytometric histograms. Thin dotted line: isotype control; shaded: CD86 unstimulated; continuous line: CD86 poly(I:C) stimulated; thick dotted line: CD86 LPS stimulated. contr., control. (H) Geometric mean fluorescence intensity (GeoMFI) of CD86 staining ± SD of n = 2 independent cultures. *, P < 0.05; **, P < 0.01 (Student’s t test). All data are representative of at least three independent experiments.
Figure 4.

Defective TLR3- and TLR4-induced activation of IRF3 in Lyst-mutant Bg-J cells. (A) WT and Bg-J BMDMs were stimulated with 10 µg/ml poly(I:C) for the indicated times. Whole cellular lysates were immunoblotted (IB) for phosphorylated IRF3 (p-IRF3), p-TBK1, or p-Erk1/2. Reprobing with anti-IRF3, TBK1, and Erk1/2, respectively, served as the loading control. The arrowhead indicates p-IRF3. (B) Whole cellular lysates from WT and Bg-J BMDMs were immunoblotted with antibodies against TRAM, TRIF, TRAF3, and Rab7. Immunoblotting with actin-specific antibodies served as a loading control. (C) WT and Bg-J BMDMs were stimulated with 100 ng/ml LPS for the indicated times. TLR4 endocytosis was measured by flow cytometric assessment of the loss of TLR4 cell-surface expression. Data are mean ± SD from duplicate cultures. (D–F) BMDMs from WT and Bg-J mice were stimulated with 100 ng/ml LPS for the indicated times. (D) Whole cellular lysates were immunoblotted for p-Erk1/2, p-p38, p-JNK, p-IκBα, and IκBα. Blots were reprobed for total Erk2, p38, and actin to verify equal protein loading. (E) Immunoblot analysis as in D using antibodies specific for p-IRF3. p-IRF3 is indicated by an arrowhead. Reprobing with anti-IRF3 served as the loading control. (F) Nuclear extracts were prepared and analyzed by immunoblotting for p-IRF3 and total IRF3. Blots were reprobed for GAPDH (cytoplasmic marker) and lamin A/C (nuclear marker). Whole cellular lysate from WT BMDMs served as a control. (G and H) WT and Bg-J BMDMs were treated with 10 µg/ml poly(I:C) or 100 ng/ml LPS or left unstimulated (unstim.). After 4 h of stimulation, cell-surface expression of CD86 was determined by flow cytometry. (G) Representative flow cytometric histograms. Thin dotted line: isotype control; shaded: CD86 unstimulated; continuous line: CD86 poly(I:C) stimulated; thick dotted line: CD86 LPS stimulated. contr., control. (H) Geometric mean fluorescence intensity (GeoMFI) of CD86 staining ± SD of n = 2 independent cultures. *, P < 0.05; **, P < 0.01 (Student’s t test). All data are representative of at least three independent experiments.

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