Fig. 4.

Activation of TLR15 is accompanied by proteolytic cleavage. (A) Immunoblot analysis of COS-7 cells expressing C-terminal Flag-tagged TLR15 treated with 100 ng mL⁻¹ proteinase K for the indicated times. The upper band of the TLR15 doublet (arrow) is proteolytically cleaved to a C-terminal 70-kDa truncated receptor (arrow with asterisk). Cleavage does not occur after inactivation of the protease by PMSF (1 mM). (B) Immunoblot analysis of TLR15 cleavage in COS-7 cells stimulated with the indicated proteases (100 ng mL⁻¹) for 45 min. (C) Stimulation of TLR15-transfected COS-7 cells with sterile culture supernatants of the yeast C. guilliermondii and P. aeruginosa (45 min) followed by immunoblot analysis of TLR15 cleavage. (D) COS-7 cells expressing C-terminal Flag-tagged chTLR15, chTLR5, hTLR4, hTLR9, or chTLR21 were stimulated with 100 ng mL⁻¹ proteinase K for 45 min and immunoblotted. (E) Proteinase K treatment of TLR15 expressed in chicken DF-1 cells resulted in an identical C-terminal receptor cleavage fragment as seen in COS-7 cells. C-terminal Flag-tagged TLR15 was detected by using M2-α-Flag.