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. Author manuscript; available in PMC: 2013 May 25.

Published in final edited form as: Circ Res. 2012 Apr 24;110(11):1435–1444. doi: 10.1161/CIRCRESAHA.112.269506

The TRPC1/4 complex was purified from pulmonary artery endothelial cells. [A] Orai1 was expressed in control cells, and was resolved in the supernatant and not the pellet fraction following lysate exposure to salt (100 mM KI). Doxycycline treatment for 72-hours decreased Orai1 protein, an effect observed at concentrations as low as 0.2 μg/mL, with maximal effect seen at 1-3 μg/mL (left panel). This effect of doxycycline was reverseable 48-hours following doxycycline withrawal (right panel). [B] FRET was observed between TRPC4 and Orai1 in control cells (data not shown). Exposure to doxycycline for 72-hours abolished the Orai1-TRPC4 FRET response. Doxycycline withdrawal resulted in a recovery of Orai1-TRPC4 FRET, consistent with the re-appearance of Orai1 in the TRPC1/4 complex. FRET efficiency is derived from the summary of a minimum of 15 separate measurements. ND refers to not detected.

The TRPC1/4 complex was purified from pulmonary artery endothelial cells. [A] Orai1 was expressed in control cells, and was resolved in the supernatant and not the pellet fraction following lysate exposure to salt (100 mM KI). Doxycycline treatment for 72-hours decreased Orai1 protein, an effect observed at concentrations as low as 0.2 μg/mL, with maximal effect seen at 1-3 μg/mL (left panel). This effect of doxycycline was reverseable 48-hours following doxycycline withrawal (right panel). [B] FRET was observed between TRPC4 and Orai1 in control cells (data not shown). Exposure to doxycycline for 72-hours abolished the Orai1-TRPC4 FRET response. Doxycycline withdrawal resulted in a recovery of Orai1-TRPC4 FRET, consistent with the re-appearance of Orai1 in the TRPC1/4 complex. FRET efficiency is derived from the summary of a minimum of 15 separate measurements. ND refers to not detected.