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. Author manuscript; available in PMC: 2019 Apr 15.
Published in final edited form as: J Immunol. 2018 Mar 12;200(8):2809–2818. doi: 10.4049/jimmunol.1700691

Fig. 7. EGFR on internal membranes, but not plasma membrane, is required for TLR9 signaling.

Fig. 7

(A) No interaction of EGFR with TLR9 in 293XL-TLR9-HA cells cultured in serum-free (starved) medium, as opposed to serum containing (normal) medium, with and without 30 min of CpG stimulation (serum starved before and during CpG treatment). (B) Lack of CpG-induced tyrosine phosphorylation of TLR9 in 293XL-TLR9-HA cells cultured in starved medium. Tyrosine phosphorylation of TLR9 was analyzed after 30 min of CpG treatment (serum starved before and during CpG treatment). (C) Serum-free (starved) condition inhibits IFNB1 and IL6 production. 293XL-TLR9-HA cells were treated with CpG under normal or serum starved (SS) conditions (serum starved before and during CpG treatment). The induction of IFNB1 mRNA and IL6 mRNA were analyzed by qRT–PCR. (D) EGFR activity is inhibited by cetuximab (Cet). Inhibition of EGF (100 ng/ml) induced EGFR tyrosine 1068 phosphorylation with different concentrations of Cet. HEK293 cells were pretreated with different concentrations of Cet for 1 h, the cells were then stimulated with EGF for 10 min in the absence or presence of Cet. The cell lysates were analyzed for EGFR pY1068, EGFR and actin. (E) No effect on gene induction by CpG by inhibiting EGFR on the cell surface. 293XL-TLR9-HA cells were pretreated with Cet (100 μg/ml) for 1h, the cells were stimulated with CpG for 6h in the absence or the presence of Cet and the induction of TNF and IFNB1 mRNA were analyzed by qRT–PCR. (F) Confocal microscopy to demonstrate co-localization of TLR9 and EGFR on early endosomal membrane. HT1080 cells expressing TLR9-YFP were used. The left three panels show the sub-cellular locations of EGFR (red), TLR9 (green) and early endosomal marker (magenta); the right three panels show their co-localization using ImageJ software Co-localization plugin where the white dots represents co-localization. Scale bars: 10 μm. Error bars were calculated as mean ± SEM from three biological replicates. P-values were calculated using two-tailed unpaired Student’s t-test; ***P < 0.001. The data are representative of at least three independent experiments.