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. Author manuscript; available in PMC: 2018 Nov 16.
Published in final edited form as: Cell Rep. 2018 May 29;23(9):2667–2677. doi: 10.1016/j.celrep.2018.04.110

Figure 1. AIBP interaction with TLR4 and selective cholesterol efflux.

Figure 1.

A, Yeast two-hybrid was performed with pB42AD-AIBP and pLexA-TLR4 ectodomain. The positive control was the yeast cell line EGY48/p80p-LacZ co-transfected with pLexA53 and pB42ADT; the negative control was the yeast cell line co-transfected with pLexA and pB42AD. B, HEK293 cells were co-transfected with the flag-tagged TLR4 ectodomain and flag-tagged AIBP. AIBP from cell lysates were pulled down with either anti-AIBP antibody, anti-TLR4 antibody or respective isotype control IgG. Blots of the pull-down or total cell lysates were probed with an anti-flag antibody. C, Peritoneal elicited macrophages from WT and Tlr4−/− mice were incubated for 2 hours on ice with 2 μg/ml BSA or 2 μg/ml AIBP (with a His-tag) and then subjected to a flow cytometry analysis with a FITC-conjugated anti-His antibody. D, BV-2 cells were stimulated with 100 ng/ml LPS for 15 min, placed on ice, and 2 μg/ml AIBP (His-tagged) or BSA were added for 2 hours, and cells were subjected to a flow cytometry analysis with a FITC-conjugated anti-His antibody. Mean±SEM; n=4; ***, p<0.001 (Student’s t-test). E, Primary brain microglia cells were loaded with 3H-cholesterol, equilibrated and then sequentially incubated with 0.2 μg/ml AIBP or BSA for 1 hour and 100 ng/ml LPS for 1 hour in complete medium. Cholesterol efflux was measured as described in Methods. Mean±SEM; n=3–5; *, p<0.05 (Student’s t-test). F, Human THP-1-derived macrophages were loaded with 3H-cholesterol, equilibrated and incubated for 24 hours with 3 μg/ml ApoA-I and 0.1% BSA, in the presence or absence of 0.2 μg/ml AIBP. LPS (10 μg/ml) was added during equilibration and efflux incubations. Mean±SEM; n=4; *, p<0.05 (Student’s t-test). G, Human THP-1-derived macrophages were loaded with acetylated LDL (acLDL; 50μg/ml) and 3H-cholesterol, equilibrated and incubated for 24 hours with 3 μg/ml ApoA-I and 0.1% BSA, in the presence or absence of 0.2 μg/ml AIBP. Cholesterol efflux was measured as described in Methods. Mean±SEM; n=4; *, p<0.05 (Student’s t-test). See also Figure S1A.