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. 2018 Apr 2;217(4):1411–1429. doi: 10.1083/jcb.201707027

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© 2018 Yurchenko et al.

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Figure 6. — SLAMF1 interacts with TRAM protein. (A) Endogenous IPs using specific anti-SLAMF1 mAbs from macrophages stimulated by LPS. (B) Endogenous IPs using anti-TRAM polyclonal antibodies from macrophages stimulated by LPS. (C) TRAM^Flag-precipitated SLAMF1 and SLAMF1ct was needed for interaction with TRAM. (D) Coprecipitation of TRAM deletion mutants: TIR domain (68–235), short TRAM TIR domain (68–176 aa), and N-terminal (1–68 aa) or C-terminal (158–235 aa) domains with SLAMF1 protein. (E) Coprecipitation of TRAM deletion mutants containing the N-terminal part of TRAM TIR domain with SLAMF1. (F) Coprecipitation of SLAMF1^Flag deletion mutants with TRAM^YFP. (G) Coprecipitation of human SLAMF1^Flag with human TRAM^YFP and of mouse SLAMF1^Flag with mouse TRAM^EGFP. Black dashed lines indicate that intervening lanes have been spliced out. (H) Human SLAMF1 cytoplasmic tail coprecipitation with TRAM^YFP with or without amino acid substitutions at 321–324. Graphs under C–F summarize the IPs’ results. Indicated constructs were transfected to HEK293T cells, and anti-Flag agarose was used for the IPs. For endogenous IPs, specific SLAMF1 or TRAM antibodies were covalently coupled to beads. At least three independent experiments were carried out for anti-Flag IPs, and five independent experiments were carried out for the endogenous IPs, and one representative experiment is shown for each. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.