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1.
Figure 8

Figure 8. From: Membrane-tethered MUC1 Mucin is Phosphorylated by EGFR in Airway Epithelial Cells and Associates with TLR5 to Inhibit Recruitment of MyD88.

Co-localization of Muc1 and TLR5 in mouse airway epithelium in vivo. Paraffin embedded mouse lung sections from uninfected (CON) and PAK intranasally-infected Muc1+/+ mice were processed for immunostaining with anti-CT2 and (A) anti-TLR5 or (B) anti-phosphotyrosine antibodies, followed by incubation with fluorescein-conjugated secondary antibodies and DAPI to counter stain nuclei. Scale bars represent 20 µm. Arrows indicate areas of co-localization.

Kosuke Kato, et al. J Immunol. ;188(4):2014-2022.
2.
Figure 4

Figure 4. From: Membrane-tethered MUC1 Mucin is Phosphorylated by EGFR in Airway Epithelial Cells and Associates with TLR5 to Inhibit Recruitment of MyD88.

Association of MUC1 and TLR5 in 293T cells. 293T cells were untransfected or transiently transfected with expression plasmids for TLR5-FLAG and/or(A) full length MUC1 or (B) MUC1ΔCT. At 24 h post-transfection, equal protein amounts of cell lysates were used for IP with isotype-matched normal mouse IgG, anti-FLAG (TLR5-FLAG), anti-CT2 (full length MUC1), or anti-MUC1 ectodomain (GP1.4)(MUC1ΔCT). IPed proteins were subjected to Western blotting with the indicated antibodies. Protein expression levels of TLR5 and MUC1 were verified in the same lysates used for IP. The results are representative of 2–3 independent experiments.

Kosuke Kato, et al. J Immunol. ;188(4):2014-2022.
3.
Figure 7

Figure 7. From: Membrane-tethered MUC1 Mucin is Phosphorylated by EGFR in Airway Epithelial Cells and Associates with TLR5 to Inhibit Recruitment of MyD88.

Pa infection increases tyrosine phosphorylation of the Muc1 CT in vivo. Muc1+/+ and Muc1−/− mice were intranasally infected with PAK (1.0 × 107 CFU/mouse). At 0, 4, and 24 h post-infection, lung homogenates were prepared and equal amounts of protein were used for IP with (A, C) anti-CT2 antibody or (B) anti-phosphotyrosine antibody. IPed proteins were subjected to Western blotting with the indicated antibodies. Each lane represents a lung homogenate from an individual mouse. The results are representative of 2–3 independent experiments.

Kosuke Kato, et al. J Immunol. ;188(4):2014-2022.
4.
Figure 9

Figure 9. From: Membrane-tethered MUC1 Mucin is Phosphorylated by EGFR in Airway Epithelial Cells and Associates with TLR5 to Inhibit Recruitment of MyD88.

Schematic illustration of the proposed mechanism through which MUC1 negatively regulates TLR5 signaling. Step 1, TLR5 on epithelial cells sense bacteria-derived flagellin. Step 2, activated TLR5 triggers MyD88-dependent signaling to induce the release of inflammatory mediators which results in recruitment of leukocytes into the site of infection to clear the bacteria. Step 3, inflammatory products, such as neutrophil elastase and TNF-α, up-regulate MUC1 expression. Step 4, EGFR activated by TLRs or other means phosphorylates the MUC1 CT at Y46. Step 5, MUC1 binds to TLR5 which interferes with the recruitment of MyD88 to TLR5.

Kosuke Kato, et al. J Immunol. ;188(4):2014-2022.
5.
Figure 5

Figure 5. From: Membrane-tethered MUC1 Mucin is Phosphorylated by EGFR in Airway Epithelial Cells and Associates with TLR5 to Inhibit Recruitment of MyD88.

Association of MUC1 and TLR5 in human airway epithelial cells. (A)Equal protein amounts of lysates from A549, MTSE, and NHBE cells were used for IP with isotype-matched normal IgG or anti-CT2 to precipitate MUC1 and IPed proteins were subjected to Western blotting with the indicated antibodies. (B) NHBE cells were untreated (CON) or treated for 48 h with heat-inactivated PAK (1.0 × 107 CFU/ml). Equal protein amounts of cell lysates were IPed as in (A) (left panel). The density of each TLR5 band that was normalized to the density of the corresponding MUC1 CT band is presented. The results are representative of 2–3 independent experiments.

Kosuke Kato, et al. J Immunol. ;188(4):2014-2022.
6.
Figure 2

Figure 2. From: Membrane-tethered MUC1 Mucin is Phosphorylated by EGFR in Airway Epithelial Cells and Associates with TLR5 to Inhibit Recruitment of MyD88.

Effect of MUC1 expression on TNF-α-induced activation of NF-κB, p38, and ERK1/2. 293-TLR5 and 293-TLR5/MUC1 cells were untreated or treated for 6 h with flagellin (1.0 µg/ml), TNF-α (100 ng/ml), or flagellin plus TNF-α. Equal protein amounts of cell lysates were subjected to (A) luciferase assay to measure NF-κB reporter activity and (B) Western blotting with the indicated antibodies. Each bar represents the mean ± SEM value (n= 3). *, P< 0.05 and †, P> 0.05 compared with untreated controls. Note the different ordinate scales in (A). The results are representative of 3 independent experiments.

Kosuke Kato, et al. J Immunol. ;188(4):2014-2022.
7.
Figure 3

Figure 3. From: Membrane-tethered MUC1 Mucin is Phosphorylated by EGFR in Airway Epithelial Cells and Associates with TLR5 to Inhibit Recruitment of MyD88.

Effect of MyD88, IRAK1, or TRAF6 overexpression on MUC1-mediated suppression of TLR5 signaling. (A, C) 293-TLR5 and 293-TLR5/MUC1 cells were untreated or treated with flagellin (1.0 µg/ml) or heat-inactivated PAK (1.0 × 107 CFU/ml) for 30 min. Equal protein amounts of cell lysates were used for IP with anti-FLAG antibody (TLR5) or isotype-matched normal mouse IgG, and IPed proteins were subjected to Western blotting with the indicated antibodies. (B) 293-TLR5 and 293-TLR5/MUC1 cells were transfected with TRAF6 (left panel), IRAK1 (middle panel), or MyD88 (right panel) expression plasmids. The cells were untreated or treated for 6 h with flagellin (1.0 µg/ml). Equal protein amounts of cell lysates were subjected to luciferase assay to measure NF-κB reporter activity. Each bar represents the mean ± SEM value (n= 3). *, P< 0.05. The results are representative of 3 independent experiments.

Kosuke Kato, et al. J Immunol. ;188(4):2014-2022.
8.
Figure 1

Figure 1. From: Membrane-tethered MUC1 Mucin is Phosphorylated by EGFR in Airway Epithelial Cells and Associates with TLR5 to Inhibit Recruitment of MyD88.

Effect of MUC1 expression on TLR5-dependent IL-8 promoter activity and activation of NF-κB, p38, and ERK1/2. (A–D) 293-pcDNA3.1, 293-TLR5, and 293-TLR5/MUC1 cells, or (E, F) Muc1−/− MEF cells transfected with pcDNA3.1 empty vector or MUC1 expression plasmid were untreated (CON) or treated for 6 h with heat-inactivated Pa strain K (PAK, 1.0 × 107 CFU/ml) or flagellin (1.0 µg/ml). Equal protein amounts of cell lysates were subjected to (A, D, F) Western blotting with the indicated antibodies, or luciferase assays for (B) IL-8 promoter activity or (C, E) NF-κB reporter activity. Each bar represents the mean ± SEM value (n= 3). *, P< 0.05 and †, P>0.05 compared with untreated controls. The results are representative of 3 independent experiments.

Kosuke Kato, et al. J Immunol. ;188(4):2014-2022.
9.
Figure 6

Figure 6. From: Membrane-tethered MUC1 Mucin is Phosphorylated by EGFR in Airway Epithelial Cells and Associates with TLR5 to Inhibit Recruitment of MyD88.

Phosphorylation of the MUC1 CT at Y46 by EGFR increases MUC1/TLR5 association.(A, C–H) A549 cells or 293T cells, transiently transfected with expression plasmids for TLR5 plus CD8/MUC1-WT or CD8/MUC1-Y46 F, were untreated or treated for 30 min with pervanadate (0.2 µM), AG1478 (100 nM) or TGF-α (100 ng/ml). Equal amounts of cell lysates were used for IP with anti-CT2, anti-CD8 (CD8/MUC1), or anti-FLAG (TLR5-FLAG), and IPed proteins were subjected to Western blotting with the indicated antibodies. (D) For ELAM-1-luciferase reporter assay, cells were untreated or treated with PAK and assayed as in Figure 1. Luciferase activity of pcDNA3.1-transfectants treated with PAK was set as 100%. Each bar represents the mean ± SEM value (n= 4). *, P< 0.01. Transfection efficiency of CD8/MUC1-WT and CD8/MUC1-Y46 F was verified by Western blotting. (B) A schematic illustration of the CD8/MUC1 chimeric protein. The results are representative of 3 independent experiments.

Kosuke Kato, et al. J Immunol. ;188(4):2014-2022.

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