Fig. 1. Hypoxia induces CD105 protein expression. HDMECs were cultured under hypoxic conditions for up to 24 hours, and CD105 expression was quantified by flow cytometry (A), immunoblotting (B) and ELISA (C) as described in Materials and Methods. (A) Hypoxic culture resulted in a maximal cell surface expression of CD105 at 16 hours (86.3% increase). The data represent five samples at each time point collected from five separate experiments (*P<0.05 and **P<0.01 compared with 0 hours as analysed by one-way ANOVA followed by the Duncan test). (B) Cell extracts were resolved on 4-7.5% SDS-PAGE under non-reducing conditions and electrophoretically transferred onto membranes. The blot was probed using mAb E9 and a rabbit anti--actin antibody. The maximal expression (as quantified on a densitometer) of CD105 was seen at 16 hours of hypoxic culture. Similar results were observed when the blots were reprobed using mAb 44G4 (data not shown). The bar chart shows the CD105 signal intensity relative to actin, pooled from three experiments. (C) Conditioned medium was collected, and soluble CD105 levels were quantified using a chemiluminescence ELISA system. CD105 levels peaked at 16 hours of hypoxic culture. The data represent six replicates at each time point pooled from three separate experiments (*P<0.05 and **P<0.01 compared with 0 hours as analyzed by one-way ANOVA followed by the Duncan test). Vertical bars indicate the standard error of the means.