Anti pkd

NRVM were pretreated with vehicle (0.1% DMSO) or CID755673 (10, and 25 μm) and stimulated with endothelin 1 (100nM). Cells were then washed three times with ice cold PBS and lysed in buffer (30 mM deoxycholate, 1M Tris, 5M NaCl, 10% SDS, 10% NP40, 500 mM NaF, 1X HALT protease inhibitor cocktail, 1X phosphatase inhibitor cocktail set IV) (Fisher, Thermo Scientific, Calbiochem). Following 1-2hr nutation, cell lysates were centrifuged at max speed for 25 min (4°C). Lysates were quantified and diluted in 6X SDS Laemmli buffer. Normalized amounts of protein lysates were resolved by SDS-PAGE and then transferred to polyvinylidene fluoride membranes (0.45 μm pore size, Millipore). Prior to antibody incubation, membranes were blocked with Odyssey Blocking Buffer (LI-COR Biosciences) to prevent non-specific binding.
Immunoblotting was performed at room temperature (1hr) or 4°C (overnight) with the following antibodies: anti-PKD-pSer916 (1:1000; Cell Signaling Technology), anti-PKD (1:1000; Cell Signaling Technology), anti-βtubulin (1:1000; Cell Signaling Technology). After 3-5 washes with TBS-T, membranes were then incubated at room temperature (1hr) with the appropriate secondary antibody (IRDye680 Donkey anti-rabbit IgG 1:20,000 LI-COR Biosciences). Bound antibody was then detected with the LI-COR Biosciences Odyssey System, and the intensity was analyzed with Image Studio Version 2.0.

All chemicals were from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise specified. [ 125 I]iodine was from Perkin Elmer (Wellesley, MA, USA). 1-Palmitoyl-2-oleoyl-sn-glycerol (POG) was from Avanti Polar Lipids (Alabaster, AL, USA). Nile red and Alexa Fluor 488 -conjugated BTX (Alexa Fluor 488 -BTX) were from Molecular Probes (Eugene, OR, USA).
Rabbit polyclonal antibodies anti-PKD, anti-pPKD (Ser 916 ), anti-pPKC␣/␤II (Thr 638/641 ) and anti-pPKC␦/ (Ser 643/676 ) were from Cell Signaling (Beverly, MA, USA). Mouse monoclonal antibodies anti-PKC␤, anti-PKC and anti-PKC␦ were from BD Biosciences (San Jose, CA, USA). Rabbit polyclonal antibody anti-pPKC (Thr 655 ) was from Life Technologies (Grand Island, NY, USA). HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).