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. 2014 Jun 11;9(6):e99214. doi: 10.1371/journal.pone.0099214

Figure 6. Rb mediates its transcriptional effects on hypoxia-inducible gene regulation through an ARNT-TRIP230-Rb complex.

Figure 6

(A) Immuno-blot of complexes precipitated using either a mouse monoclonal antibody directed to TRIP230 or mouse control IgG from the nuclear extracts of MCF7 cells. Blots were probed for the presence of TRIP30, ARNT and Rb. The left hand lane of each blot contains nuclear extract representing 10% of input. (B) GST-ARNT-PAS-B is capable of pulling down TRIP230 and Rb. Immuno-blot analysis of GST-ARNT-PAS-B pull-down and GST pull-down of TRIP230 and Rb from MCF7 nuclear extracts. GST moieties were fixed to glutathione-agarose beads and incubated for 90 min at 4 degrees C with 500 µg of MCF7 cell nuclear extract. Input lanes were loaded with 250 µg of nuclear extract. Complete blots for panels A and B can be found in Supplemental Figure S1. GST-ARNT-PAS-B is capable of pulling down phosphorylated Rb. (C) Immuno-blot analysis of GST-ARNT-PAS-B pull-down and GST pull-down of Rb-phospho-serine780 (Rb-pS780) from MCF7 nuclear extracts harvested from cells left at normoxia (N) or treated with 1% O2 for 6 h (H). (D) Chromatin immuno-precipitation assay of EPO enhancer and VEGF promoter regions in MCF7 cells using control or Rb-phospho-serine780 antibodies. Cells were treated as described above. Rb attenuates TRIP230-mediated co-activation of ARNT-dependent transcriptional activity. The Rb- and ARNT-interaction domains are indicated. Hepa-1c1c7 cells (E) and MCF7 cells (F) were transfected with a hypoxia responsive 4xHRE-driven luciferase construct as a reporter, expression plasmids for TRIP230, TRIP230ΔRB and Rb as indicated and subjected to 1% O2 or atmospheric (20%) O2 for 24 h. Whole cell lysates were assayed for luciferase activity. (G) A schematic of the TRIP230ΔRB mutant. (H) TRIP230 protein levels are unaffected by transfection of Rb expression plasmid into Hepa1c1c7 cells. Whole cell lysates were analyzed by immuno-blot and membranes were probed with affinity-purified antibodies to TRIP230, Rb and α-tubulin. *p<0.05.