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. 2015 Dec 30;113(2):E165–E171. doi: 10.1073/pnas.1518615113

Fig. 1.

Fig. 1.

NTHi induces lysine 63 (K63)-linked polyubiquitination of MyD88 in vitro and in vivo. (A) Myc-tagged full-length of MyD88 (Myc-MyD88 WT) was cotransfected into HeLa CCL2 cells with a HA-tagged ubiquitin with all lysine residues (HA-WT Ub). Cells were treated with NTHi for the indicated time periods. Cell lysates were then immunoprecipitated (IP) with anti-HA antibody and immunoblotting (IB) was then performed with the anti-Myc antibody. (B) HeLa CCL2 cells were treated with NTHi for 1, 2, 4, or 6 h, and cell lysates were immunoprecipitated with anti-Ub antibody and analyzed by immunoblotting with anti-MyD88 antibody. (C) Anesthetized C57BL/6 mice were intratracheally inoculated with NTHi for 1, 2, 4, or 6 h. Lung lysates from mice were immunoprecipitated with anti-Ub antibody and analyzed by immunoblotting with anti-MyD88 antibody. Lysates of HeLa CCL2 (D) and mouse lung (E) were immunoprecipitated with anti-lysine 63 Ubiquitin (K63 Ub) antibody and analyzed with anti-MyD88 antibody. All data are representative of at least three independent experiments.