Human IgG1 Fc region was integrated into AccI site of pDisplay (Invitrogen #V660-20). The ectodomain of Clec2, Mincle, Clec9a, MGL-1, SIGNR1, SIGNR3, DCAR, Clec5a, DC-SIGN (human), TREM1, TREM2, TREM3, LMIR2, LMIR4, LMIR5, LMIR7, LMIR7, and LMIR8 (all from mouse origin except for DC-SIGN) were PCR amplified with the primers in Supplementary Table 1 using cDNA clones (purchased from DNAFORM) as templates, and were inserted into the upstream of the human IgG1 Fc region in-frame in pDisplay. pDisplay-mouse Dectin-1-Fc (kindly provided by Prof. Naohito Ohno, Tokyo University of Pharmacy and Life Sciences) and pME18S-mouse Dectin-2-Fc80 were used for the following Dectin-1-Fc and Dectin-2-fc expression, respectively. The plasmids were transfected into Freestyle 293 cells (Thermo Fisher Scientific #R79007) using Freestyle MAX reagent (Invitrogen #16447-100), and then cultured according to the manufacturer’s instruction. The culture supernatants were collected and then concentrated ~20 times by using VIVASPIN 20 MWCO 30 kDa (Sartorius Stedim Biotech #VS2022). These concentrated supernatants were used in the screening for proteins binding to mycobacteria. For the plate-coated lipid binding assay, TREM2- and Mincle-Fc fusion proteins were purified by using protein G columns, as descried previously80.

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