Figure 5: Tetramerization of tRXRα prevents its interaction with p85α. | Nature Communications

Figure 5: Tetramerization of tRXRα prevents its interaction with p85α.

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

Figure 5

(a) AF2/H12 is required for binding p85α. HEK293T cells transfected with HA-p85α and Myc-tRXRα or mutants were treated with TNFα (10 ng ml−1) for 1 h, and analysed by coIP assay using anti-Myc antibody. (b) Regulation of tRXRα interaction with p85α by ligand and RXRα-A/B region. HEK293T cells transfected with the indicated HA-p85α-ΔiSH2, Myc-tRXRα and RXRα-A/B were treated with or without 9-cis-RA (10−7M) for 6 h, and analysed by coIP assay using anti-Myc antibody. (c) Tetramerization of tRXRα/R316E and tRXRα/F313A was analysed in MCF-7 cells transfected with tRXRα/R316E or tRXRα/F313A, which were then treated with 9-cis-RA (10−7 M) or K-80003 (5 × 10−6 M) for 6 h. Cell lysates were subjected to BS3 crosslinking and analysed by western blotting using ΔN197 anti-RXRα antibody. (d) Interaction of tRXRα/R316E and tRXRα/F313A with p85α. HEK293T cells transfected with the indicated expression plasmids were treated with TNFα (10 ng ml−1) or 9-cis-RA (10−7M) for 1 h, and analysed by coIP assay using anti-Myc antibody. (e) Characterization of p85α interaction with tRXRα/R316E or tRXRα/F313A. HEK293T cells transfected with the indicated expression plasmids were treated with 9-cis-RA (10−7M) or K-80003 (5 × 10−6M) for 6 h., and analysed by coIP assay using anti-HA antibody. (f) Mutation of R316 impairs tRXRα cytoplasmic localization. HEK293T cells cotransfected with Myc-tRXRα/R316E and HA-p85α were pretreated with K-80003 (5 × 10−6M) for 3 h before exposed to TNFα (10 ng ml−1) for 30 min. Cells were immunostained with anti-Myc and anti-p85α antibody, and visualized by confocal microscopy. Scale bar, 10 μm. (g) Tetramerization of tRXRα impairs its interaction with p85α. HepG2 cells transfected with Myc-tRXRα together with HA-p85α-ΔiSH2 were treated with K-80003 and or 9-cis-RA. Cell lysates were then subjected to BS3 crosslinking, and analysed by western blotting using anti-Myc antibody. (h) Tetramerization of RXRα-LBD impairs its interaction with p85α-BCR. HEK293T cells transfected with Myc-RXRα-LBD together with HA-p85α-BCR were treated with K-80003. Cell lysates were subjected to BS3 crosslinking, and analysed by western blotting using anti-HA antibody. For western blotting, one of three or four similar experiments is shown.

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