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. 2014 Jun 4;13(11):5185–5197. doi: 10.1021/pr5002466

Figure 2.

Figure 2

Global comparison of the phosphorylation events that resulted from different ligand stimulation. (A) Overlap of phosphorylation sites from the two independent experiments for each ligand stimulation. Depicted are the phosphorylation sites that were quantified relative to unstimulated macrophages. The downstream bioinformatics analyses used only the reproducibly identified phosphorylation sites. (B) Distribution of phosphorylated amino acids. The total number of quantified phospho-serine (white), phospho-threonine (gray), and phospho-tyrosine (black) sites for each ligand stimulation is indicated. (C) Distribution of the phosphorylated proteins within cellular compartments. The number of proteins within each cellular compartment (black) was compared to the number of phosphoproteins obtained from each ligand stimulation (red: LPS, blue: P3C, purple: R848). Significantly over-represented GO terms are marked with asterisks, and under-represented GO terms are marked with a hash. (D) The absence of TLR4 expression affects cellular migration. C57 derived macrophages or TLR4 knock out stable transfectants in a C57 derived macrophages background were assayed in the presence or absence of 100 ng/mL LPS. Four different cell combinations were used; C57 cells were seeded in the microchamber in the presence of C57 (C57/C57), or C57 TLR4–/– cells (C57/TLR4–/–) in the well, or C57 TLR4–/– cells were seeded in the microchamber in the presence of C57 (TLR4–/–/C57) or C57 TLR4–/– (TLR4–/–/TLR4–/–) cells in the well. Cell migration was allowed to proceed for 12 h at 37 °C, and cells that transmigrated across the membrane were counted; *p < 0.005 when compared to the wild type C57 derived macrophages. (E) Delay in phosphorylation of phosphoproteins involved in the MyD88 dependent pathway (ERK1, AP-1), which responded earlier to the LPS stimuli compared to the MyD88 independent pathway (TAK1, IRF3), which is activated after TLR4 internalization. NFKBIB is an inhibitor of the MyD88 dependent pathway, and its phosphorylation event coincided with the start of the MyD88 independent pathway. The dotted lines represent significance thresholds (log fold change = ± 0.114). Experiments were performed in duplicate.