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. 2013 Nov 21;289(2):1079–1091. doi: 10.1074/jbc.M113.491522

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© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Interaction between SMILE and LXRα. A, endogenous interaction between LXRα and SMILE. Protein extracts from HepG2 cells were subject to co-immunoprecipitated (IP) with LXRα or SMILE and the interaction between LXRα and SMILE was determined by Western blotting using the SMILE antibody (left panel) or LXRα antibody (right panel). LXRα and SMILE expression (lower two panels) from the 10% lysate was analyzed by Western blotting (WB) with the indicated antibodies. B, in vitro GST pulldown assay. ³⁵S radiolabeled LXRα protein was incubated with GST-only or GST-SMILE fusion proteins. The input lane represents 10% of total volume of in vitro translated proteins used for binding assay. Protein interactions were detected via autoradiography. C, HepG2 cells were co-transfected with expression vectors for HA-LXRα with pEBG-SMILE (GST-SMILE) or pEBG alone (GST-only) and then treated with dimethyl sulfoxide or T7. Complex formation (upper two panels, GST purification) and the amount of HA-LXRα (lower panel, lysate) used for the in vivo binding assay determined the interaction with the anti-HA antibody. The same blot was stripped and re-probed with an anti-GST antibody (middle panel) to confirm expression levels of the GST fusion protein (GST-SMILE) and the GST control (GST). D, schematic representation of the structures of the LXRα deletion mutants. AB, N-terminal domain; C, DNA binding domain; DE, hinge and ligand binding domain; AF2, activation function-2 domain; Δ, deletion region. E, HepG2 cells were co-transfected with expression vectors for HA-SMILE and indicated pEBG-LXRα (GST-LXRα). The interaction was determined via Western blot using anti-HA. The same blot was stripped and reprobed with anti-GST antibody to confirm the expression levels of the GST fusion protein (GST-LXRα mutants) and the GST control (GST-only). F, effects of SMILE LXXLL mutants on LXRα-mediated transcriptional activity. HepG2 cells were cotransfected with reporter vector LXRE-luc, together with indicated expression vector for LXRα, wild-type (WT) FLAG-SMILE or FLAG-SMILE LXXLL mutants (m1, m2, m3, m4, and m5). Luciferase activity was measured 48 h after transfection.