Fig. 5

Hypoxia-induced up-regulation of sFLT1 protein secretion in 5azadC-treated choriocarcinoma cells. Three choriocarcinoma cell lines were incubated in the presence of 0.1% DMSO (vehicle control) or 10 μM 5azadC. Culture media were changed daily. After 5 days of culture, culture media were changed to fresh growth media without 5azadC and then cells were incubated for 24 h under normoxic or hypoxic conditions. Conditioned media were collected for ELISA and Western blotting. a The mRNA expression levels of three FLT1 splice variants in the three choriocarcinoma cell lines. Results are expressed as a ratio relative to the expression of β-actin mRNA. b Measurement of sFLT1 secreted from the three choriocarcinoma cell lines. The amount of sFLT1 in the conditioned media was measured by ELISA, and then normalized to the protein content of each cell. c Immunoprecipitation of FLT1 isoforms from the cell lysates derived from the three 5azadC-treated cell lines under normoxic or hypoxic conditions. Numbers represent the amount of protein subjected to immunoprecipitation. The indicated percentage of cell lysate was used as the input. d Western blotting of sFLT1 proteins secreted into the conditioned media. Numbers indicate the volume of media subjected to heparin-affinity pull-down. Uncropped images of Western blots are presented in Additional file 1: Figure S6. All values represent the mean ± SD (n = 3). Asterisks indicate a significant difference (P < 0.05). ND: not detected