Figure 2
Basal rate of carbonic anhydrase IX (CA IX) ectodomain release from various cell lines under normoxia and hypoxia. (A) Confluent monolayers of CA IX-expressing cells were washed, fresh medium was added and the shedding was allowed to proceed over a 48-h period. The media were collected, the cells were extracted and the content of CA IX was determined by ELISA with V/10 as a capture and biotinylated M75 followed by streptavidin–peroxidase as a detector. (B) Serial dilutions of the extracts and media were examined by ELISA to relate the cellular and extracellular CA IX concentrations. (C) The parallel cultures were maintained for 48 h in normoxia and hypoxia (2% O2). Harvested media (diluted 1:2) and cell extracts (diluted 1:20) were assayed in ELISA and the hypoxia-induced changes of CA IX levels in the cells and released to medium were illustrated as a ratio between the absorbance values measured in the hypoxic samples and the values of the corresponding normoxic samples. *P<0.01 and **P<0.001 by Student's t-test.
