P582S and A588T mutations do not affect HIF-1α transcriptional activity, localization and expression.

<p>HIF-1α transcriptional activity was determined 24 h after co-transfection of Saos-2 (A) and HEK293 cells (B) with plasmids expressing the indicated proteins together with the pGL3–5HRE-VEGF reporter plasmid. Values (as relative luminescence units) were determined as a ratio of firefly luciferase activity over Renilla luciferase activity and represent the mean of four different experiments performed in triplicate (+/- S.E.). Statistical differences were assessed using unpaired-t-test. P values>0.05 were considered as statistically non-significant (ns). Saos-2 (C) and HEK293 cells (D) where transfected with plasmids expressing the indicated proteins and their localization was observed after 24 h, with immunofluoresence, using an anti-FLAG antibody. Protein levels of FLAG-HIF-1α wt and mutants were determined in Saos-2 (E) and HEK293 (F) cells after 24-48 h post-transfection, in Western blot using a monoclonal anti-HIF-1α antibody and anti-tubulin as loading control. Densitometric analysis of the bands was performed with the public domain software for image analysis ‘ImageJ’. FLAG-HIF-1α quantities were normalized against corresponding tubulin and expressed as fold increase against wt FLAG-HIF-1α (100%).