The second method used the Innozyme TACE Activity KitTM (EMD during Biosciences). Serum starved R-VSMCs in 10-cm culture dishes were incubated in the presence or absence of 20 nm human plasma KK, lysed as recommended by the manufacturer, and normalized for protein content. Equal amounts of sample were loaded into 96-well plates precoated with an anti-human TACE monoclonal antibody, incubated for 2 h at room temperature, and washed, after which the activity of immobilized ADAM17 was measured using MCA-KPLGL-Dpa-AR-NH2 as above. Protein Immunoblotting Endogenous phosphoproteins were detected by immunoblotting whole cell lysates using phosphorylation site-specific IgG.
Serum-starved R-VSMC in six-well plates were stimulated at 37 ��C as described in the figure legends, lysed with 1�� hypotonic sample buffer (Invitrogen), sonicated, boiled, resolved by 4�C12% gradient SDS-PAGE (Invitrogen), and transferred to polyvinylidine difluoride membrane (Millipore, Billerica, MA). Phosphorylated ERK1/2 was detected using rabbit polyclonal anti-phospho-ERK1/2 IgG (Cell Signaling Technology, Beverly, MA) with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Phosphorylated JNK1/2 was detected using rabbit polyclonal antiphospho-JNK1/2 IgG (Cell Signaling Technology). Phosphorylated EGF receptor was detected using rabbit polyclonal anti-ErbB1 phospho-Tyr1068 IgG (Cell Signaling Technology). Total ERK1/2, measured to confirm equal loading of whole cell lysate samples, was detected using polyclonal anti-ERK1/2 IgG (Upstate Biotechnology, Waltham, MA).
Immune complexes were visualized on x-ray film by enzyme-linked chemiluminescence. Multiple exposures were made of each immunoblot and band intensities on optimally exposed films were quantified using a Fluor-S MultiImager (Bio-Rad). ADAM17 Ligand Shedding H-VSMCs at 90% confluence in 10-cm culture dishes were serum-starved for 24 h, after which the medium was replaced with 7 ml of medium containing 0.5% fetal bovine serum and monolayers were incubated for 3 h, with or without 20 nm human plasma KK. Conditioned medium was collected and concentrated using iCONTM Concentrator 7ml/9K tubes (Pierce) centrifuged at 4000 rpm at 4 ��C for 1 h. Aliquots of concentrated medium were mixed with 2�� sample buffer and resolved by SDS-PAGE.
Amphiregulin and TNF-�� release were quantified by immunoblotting using rabbit polyclonal antiamphiregulin (Santa Anacetrapib Cruz Biotechnology) or anti-TNF�� IgG (Chemicon International, Inc., Billerica, MA) with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody. Confocal Microscopy To visualize GFP-PAR1�C4 internalization, HEK293 cells were transfected with plasmids encoding GFP-tagged PAR1, PAR2, or PAR4 as described.