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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: Arch Biochem Biophys. 2012 Mar 23;521(1-2):90–94. doi: 10.1016/j.abb.2012.03.017

Figure 2.

Figure 2

Figure 2

Effect of acetazolamide on CAIX activity in intact MDA-MB-231 cells - MDA-MB-231 cells were grown and exposed to hypoxia as described in Figure 1. Panel A: Cells were harvested using either trypsin (T) treatment (0.25%, 3 min at 37°C) or enzyme-free cell dissociation (CD) buffer (5 min at 37°C). Cells were collected and lysed. Western blotting was performed to detect the presence of CAIX and CAII. Two independent experiments are shown. Panel B: Cells harvested using Cell Release Buffer were assayed for carbonic anhydrase activity by 18O exchange in the absence or presence of 1 μM acetazolamide at pH 7.4, 16°C. Each progress curve was generated with 5 × 105 cells/mL. We also allowed the cells to settle to test for activity in the medium that overlaid the cells (supernatant: uncatalyzed reaction). Atom fractions of 18O in CO2 were continuously collected. For ease of illustration, data points at 25 second intervals are shown. Data are representative of at least three independent experiments.