Figure 2
B7-H4 suppresses RCOR2 to reduce RUNX1 expression through PTEN/AKT signaling. (a) Protein levels of RUNX1 and RCOR2 were evaluated in Rcor2-over-expressing C1498 cells (first panel), Rcor2-knockdown C1498 cells (second panel) and WT and B7-H4-null BM leukemia cells (third panel) using immunoblotting. (b) Expression levels of p-AKT, AKT and HIF-1α were measured in the WT and B7-H4-null BM AML cells (first panel). BM AML cells were treated with the AKT inhibitor MK2206 at the indicated doses (0, 2.5 and 5 μM) for 72 h followed by the evaluation of the protein levels of p-AKT, AKT, HIF-1α and RCOR2 using immunoblotting (second panel). Luciferase activity was measured in 293T cells transfected with a luciferase reporter containing the Rcor2 promoter (−1770–719) and indicated doses of HIF-1α plasmid, and normalized by Renilla. The relative luciferase activity of HIF-1α were further standardized to that of control vector (n=6, third panel). (c) Protein levels of PTEN were measured in WT and B7-H4-null AML cells using immunoblotting (first panel). StrepII-tagged B7-H4 and Flag-tagged (a specific tag with eight amino acids for detection) PTEN were overexpressed in 293T cells, and their lysates were co-immunoprecipitated using strep-tactin sepharose followed by western blotting analysis for Flag (PTEN) and endogenous levels of AKT (second panel). StrepII-tagged B7-H4 and HA-tagged AKT were overexpressed in 293T cells, and their lysates were co-immunoprecipitated (IP) using strep-tactin sepharose followed by western blotting analysis for HA (AKT) and endogenous levels of PTEN (third panel). StrepII-tagged B7-H4, Flag-tagged PTEN and HA-tagged ubiquitin were overexpressed in 293T cells followed by the addition of the proteasome inhibitor MG132 in culture during the last 4 h. The lysates were immunoprecipitated using an anti-Flag M2 affinity gel followed by western blotting for HA (ubiquitin) (fourth panel).
