Figure 1
From: Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2
Expression of endosialin in response to hypoxia. (A) FIB-3 and 42-MG-BA cells were incubated for 24 h in normoxic or hypoxic conditions. Total RNA was extracted and expression of endosialin mRNA was analysed by quantitative RT–PCR and normalized to β-actin levels. Vascular endothelial growth factor (VEGF) mRNA was used as a positive control. (B) Specificity of the monoclonal antibody (MAb) M78 for endosialin protein was evaluated by immunoblotting using 42-MG-BA cells that naturally express endosialin and HeLa cells transfected with endosialin cDNA (HeLa-endosialin). Mock-transfected HeLa cells served as a negative control. M78 MAb could recognise both the non-glycosylated core protein (95 kDa) and the highly glycosylated endosialin (165 kDa). (C) Immunoblotting analysis of endosialin protein levels in FIB-3 (50 μg total proteins/lane) and 42-MG-BA cells (80 μg total proteins/lane loaded due to lower endosialin expression level, see part a). The cells were incubated for 24 h in hypoxia (2% oxygen, +) or normoxia (−). M78 MAb revealed increased expression of endosialin in the hypoxic cells. α-Tubulin antibody was used for a loading control.
