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. 2021 Dec 13;39(1):msab354. doi: 10.1093/molbev/msab354

Fig. 7.

Fig. 7.

Full-length Eudyptes consensus TLR15 is nonfunctional, and is therefore a cryptic pseudogene. (A) Expression and stimulation of TLR15 from chicken (ch), Northern fulmar (Fgl), Emperor penguin (Afo), and a consensus intact Eudyptes TLR15 (Eud) in HEK-Blue Null1 NF-κB reporter cells. Cells expressing TLR15 constructs, or empty vector (EV) control, were challenged with brewer’s yeast (S. cerevisiae) or A. fumigatus lysates, or untreated (UT; medium control). (B) Immunoblot expression analysis of Eudyptes TLR15 in HEK-Blue Null1 NF-κB reporter cells with the same treatments as (A). (C) Schematic diagram of Eudyptes TLR15 chimaeras generated to functionally test the extracellular domain and intracellular domain independently. Eudyptes TLR15 TIR domain was attached to the extracellular domain from murine CD4 (muCD4-EudTIR15), and Eudyptes TLR15 extracellular domain was attached to the TIR domain from chicken TLR15 (EudTLR15-chTIR15). Equivalent chimaeric constructs were made for muCD4-AfoTIR15 and muCD4-FglTIR15. (D) NF-κB response to expression of muCD4-TIR domain chimaeras (and no additional stimulation). (E) NF-κB response to expression of Eud-chTIR15 or chTLR15 and stimulation with S. cerevisiae or A. fumigatus lysates. (F) Immunoblot expression analysis of EudTLR15-chTIR15 in HEK-Blue Null1 cells. CMV14 and pT (pTarget) refer to the expression vectors used for each construct. Statistical significance was considered to be P < 0.05 and “***” denotes P < 1 × 10−7 following Dunnett’s procedure for multiple comparisons; NS, not significant.