Intracellular calcium levels were measured either with using calcium indicator Fluo-4 AM (Molecular Probes) according to the manufacturer’s instructions or with the calcium-activated photoprotein, aequorin, as described previously (15 (link)). In brief, for aequorin-mediated fluorescence measurements COS-7, HULEC5α or Cav-1 KO HULEC5α cells were transduced with adenoviruses encoding aequorin, RFP or Cav-1 at 15 MOI, and 24 h later the aequorin was reconstituted by treating cells with 5 μM coelenterazine (Sigma, St. Louis, MO) for 1 hour in serum-free and phenol free DMEM containing 0.1 mM EDTA. Then, the cells were exposed to different concentrations of PLY in Hank’s Balanced Soult Solution (HBSS) for 10 minutes, and luminescence was recorded using a PolarSTAR luminometer (BMG Labtech, Cary, NC). After the baseline luminescence values were recorded, calcium chloride was injected at a 1.8 mM final concentration to enable the measurement of calcium induced changes in luminescence which reflect calcium influx.
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