Western Blotting. After treatment as outlined for individual experiments, EC were subsequently washed with cold (4�� C) Ca2+/Mg-free PBS and lysed with 0.3% SDS lysis buffer containing protease inhibitors (1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 0.2 trypsin inhibitor unit/ml aprotinin, 10 ��M leupeptin, different and 5 ��M pepstatin A). Sample proteins were separated with 4 to 15% SDS-PAGE gels (Bio-Rad, Hercules, CA) and transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore Corporation). Membranes were then immunoblotted with primary antibodies (1:500�C1000, 4��C, overnight) followed by secondary antibodies conjugated to horseradish peroxidase (1:5000, room temperature, 30 min) and detected with enhanced chemiluminescence (Pierce ECL or SuperSignal West Dura; Pierce Biotechnology, Rockford, IL) on Biomax MR film (Carestream Health, Rochester, NY).

Measurement of Intracellular Calcium. Measurements of [Ca2+]c using Fura-2 were performed as described previously (Harbeck et al., 2006). HPAEC plated on 25-mm glass coverslips were loaded with 1 ��M Fura-2/acetoxymethyl ester ( Invitrogen, Carlsbad, CA) for 20 min at 37��C in KRBH5 buffer (Krebs-Ringer-bicarbonate solution containing 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 1 mM KH2PO4, 25 mM NaHCO3, 10 mM HEPES-NaOH (pH 7.4), and 5 mM glucose). After replacement of the Fura-2 loading buffer with fresh KRBH5, coverslips were placed into the specimen stage of an inverted fluorescence microscope (Nikon TE-2000U). A Nikon Super Fluor 10�� objective was used for these studies.

Filters (340- and 380-nm excitation and 530-nm emission) were used for Fura-2 dual excitation ratio imaging. Imaging data acquisition and analysis were accomplished using MetaMorph/MetaFluor software (Molecular Devices, Sunnyvale, CA) and OriginPro 7E (OriginLab Corp, Northampton, MA). Fura-2 340/380 dual excitation ratios were converted to [Ca2+] by in situ calibration. To calibrate Fura-2 ratios, Rmax was obtained by treating cells with 10 ��M ionomycin and 2.5 mM Ca2+, and Rmin was obtained by treating cells with EGTA to a final concentration of 10 mM. Fura-2 ratios were converted to [Ca2+] as described by Grynkiewicz et al.

(1985) using the equation: [Ca2+] = (Kd��[(R ? Rmin)/(Rmax ? R)] �� Sf/Sb), where Kd�� is the dissociation constant for Fura-2 in the cytosol (225 nM) and Sf and Sb are the measured emission intensities at 380 nm for Ca2+-free and Ca2+-bound Fura-2, respectively. Data summaries for all Ca2+ measurements are expressed as the means �� S.E. Animals Housing and Procedures. All experiments and animal care procedures were approved by the Chicago University Animal Resource Center and were handled according to the Animal Carfilzomib Care and Use Committee Guidelines at the University of Chicago.