Fig. 5.

RASSF1A interacts with HIF1α. a Human PASMCs were transfected with si-RASSF1 and 48 h after transfection, pre-treated with indicated concentrations of MG132 for 30 min, followed by 6 h hypoxia exposure. Cell lysates were subjected to a, upper western blotting for indicated proteins, followed by a, lower densitometric quantification of relative HIF1A expression. *P < 0.05 compared to si-Control (hypoxia), one-way ANOVA followed by SNK multiple comparison test. Data represent mean ± s.e.m. n = 3 independent experiments from 3 biological replicates. b, c HEK293 cells were transfected with plasmids indicated on top of lanes. 24 h after transfection, cells were treated with 25 μM MG132, followed by 5 h hypoxia exposure. HIF1α (HIF1A) was immunoprecipitated (IP), followed by western blotting for b hydroxyl proline (Pro-OH) or c lys48 ubiquitin (K48ubi) antibody. d HEK293 cells were transfected with plasmids indicated on top of lanes and exposed to hypoxia for 6 h, followed by PHD2 IP and western blotting for indicated proteins. e HEK293 cells were transfected with plasmids indicated on top of lanes and exposed to hypoxia for 24 h. HIF1A IP and RASSF1A co-IP were detected by western blotting. f Human PASMCs were exposed to hypoxia for 24 h followed by f HIF1A IP and RASSF1A western blotting and g proximity ligation assay with HIF1A and RASSF1A antibodies. Each red spot represents for a single interaction between HIF1A and RASSF1A and DNA was stained with DAPI (blue). Scale bar: 50 μm. n = 2–3 independent experiments