Figure 1. RXR and transactivation are required for LXR-dependent inflammatory repression.

(A) Bone marrow-derived macrophages from wild-type mice were transfected with siRNA targeting RXRα and RXRβ (siRXRαβ or control (siCtrl) for 48 hr, pretreated with GW3965 (1 µM) overnight, and then stimulated with LPS (10 ng/ml) for 4 hr. (B) Immortalized MEFs from Lxrα−/−Lxrβ−/− mice reconstituted with wild-type human LXRα, AF1-deletion mutant (∆AF1), DNA-binding domain deletion mutant (∆DBD), ligand-binging domain deletion mutant (∆LBD), AF2-deletion mutant (∆AF2) or control mock were pretreated with the LXR agonist GW3965 (1 µM) overnight, followed by stimulation with LPS (10 ng/ml) for 4 hr. (C) Immortalized MEFs from Lxrα−/−Lxrβ−/− mice reconstituted with wild-type human LXRα, L439A/E441A mutant or control mock were pretreated with the LXR agonist GW3965 (1 µM) overnight, followed by stimulation with LPS (10 ng/ml) for 4 hr. Gene expression was analyzed by real-time PCR. N = 4 per group. *p < 0.05, **p < 0.01, NS, not significant. Error bars represent means ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.08009.003

Figure 1—figure supplement 1. Effects of RXR knockdown on LXR-mediated inflammatory repression.

(A, B) Bone marrow-derived macrophages from wild-type mice were transfected with siRNA targeting RXRα and RXRβ (siRXRαβ or control (siCtrl) for 48 hr, pretreated with GW3965 (1 µM) overnight, and then stimulated with vehicle or LPS (10 ng/ml) for 4 hr as indicated. Gene expression was analyzed by real-time PCR. N = 4 per group. (C) Immunoblot analysis of LXRα and LXRβ protein in HepG2 cells treated with vehicle or GW3965 (2 µM) overnight and immortalized MEFs from Lxrα−/−Lxrβ−/− mice reconstituted with wild-type human LXRα, human LXRβ or control mock. (D) Immortalized MEFs from Lxrα−/−Lxrβ−/− mice reconstituted with wild-type human LXRα and LXRβ were treated with the LXR agonist GW3965 (1 µM) overnight. Gene expression was analyzed by real-time PCR. N = 4 per group. *p < 0.05, **p < 0.01, NS, not significant. Error bars represent means ± SEM. (E) Immunoblot analysis of LXR proteins in immortalized MEFs from Lxrα−/−Lxrβ−/− mice reconstituted with wild-type human LXRs or indicated mutants. Note, the epitope recognized by the LXRα antibody is in the DBD and that recognized by the LXRβ antibody is in the AF-1 domain. Therefore, these respective deletion mutants are not detected.

DOI: http://dx.doi.org/10.7554/eLife.08009.004