Figure 3: Induction of RXRα tetramerization by K-80003 and its regulation by the N-C intramolecular interaction. | Nature Communications

Figure 3: Induction of RXRα tetramerization by K-80003 and its regulation by the N-C intramolecular interaction.

From: Modulation of nongenomic activation of PI3K signalling by tetramerization of N-terminally-cleaved RXRα

Figure 3

(a) Equal amounts of purified RXRα-LBD or mutant protein were incubated with DMSO, 9-cis-RA, and/or K-80003, and separated by non-denaturing polyacrylamide gel electrophoresis followed by Coomassie Bright Blue staining. The percentage of tetramer and dimer of RXRα-LBD or mutants was quantitated by densitometric analysis of the corresponding blots. One of four similar experiments is shown. (b) RXRα-LBD incubated with K-80003 or 9-cis-RA was subject to gel filtration chromatogram assay. Results showed that 9-cis-RA-induced RXRα-LBD was mostly in dimer (D), while K-80003-induced RXRα-LBD was mostly in tetramer (T). One of three similar experiments is shown. (c) HepG2 cells transfected with RXRα, tRXRα or RXRα-LBD were treated with 9-cis-RA or K-80003. Cell lysates prepared were then subjected to BS3 crosslinking, and analysed by western blotting using ΔN197 anti-RXRα antibody. One of more than five similar experiments is shown. (d) Schematic representations of RXRα and mutants. A/B, C, D, E/F domains in RXRα are indicated. (e) HepG2 cells transfected with HA-RXRα-A/B and Myc-RXRα-LBD were treated with 9-cis-RA, and analysed by coIP with anti-HA antibody. One of two similar experiments is shown. (f) Inhibition of K-80003-induced tRXRα tetramerization by A/B domain. HEK293T cells transfected with tRXRα together with RXRα-A/B were treated with K-80003. Cell lysates were subjected to BS3 crosslinking, and analysed by western blotting using ΔN197 anti-RXRα antibody. One of three similar experiments is shown. (g) RXRα-A/B interaction with RXRα N-terminal deletion mutants. HA-RXRα-A/B and Myc-tagged RXRα N-terminal deletion mutants were transfected in to HEK293T cells, and their interaction was analysed by coIP. (h) Interaction of RXRα-A/B with RXRα mutants. HA-RXRα-A/B and Myc-tagged RXRα-mutants were transfected together in to HEK293T cells, and their interaction was analysed by coIP. One of three similar experiments is shown. (i) Mutation of Trp305 does not affect N/C interaction. Myc-tagged RXRα-LBD or RXRα-LBD/W305Q was transfected together with HA-RXRα-A/B into HEK293T cells in the presence or absence of 9-cis-RA (10−7M). Cell lysates were prepared and analysed by coIP.

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