Pkcζ sc 216

Protein samples were subjected to sodium-dodecyl-sulfate polyacrylamide gel electrophoresis using Novex Tris–Glycine gels (8–16 %) and transferred to polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA, USA). For assessment of cPLA₂, 80 μg protein per sample was used, whereas 50 μg protein was used for PKC isoforms. Each sample represented a single animal. Membranes were probed with antibodies for the following proteins: PKCε (610086), PKCδ (610397), (BD Biosciences, San Jose, CA, USA), cPLA₂ (sc-454), p-cPLA₂ (ser505) (sc-34391) and PKCζ (sc-216) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for simultaneous detection of PKMζ and PKCι/λ. All primary blots were subsequently re-exposed to a second primary antibody directed against β-actin (MAB1501, Millipore) to verify equivalent protein loading and transfer. Samples were run in duplicate or triplicate and averaged. All bands were detected by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ, USA) and exposed to autoradiography film under non-saturating conditions. Data were quantified using NIH Image J.

The following antibodies were used in this study: GM130 (mouse; 610822; BD Transduction Laboratories), DPPIV (CD26, clone OX-61; mouse; MCA924R: Bio-Rad), ezrin (rabbit; ab41672, Abcam), P-MLC2 (Phospho-Myosin Light Chain 2; rabbit; 3674; Cell Signaling), PKC ζ (sc-216; Santa Cruz Biotechnology, Inc.), TGN38 (provided by D. Shields, Albert Einstein College of Medicine, New York), RhoA (rabbit; ab86297; Abcam), VSVG cytoplasmic domain (clone P4D5; hybridoma provided by E. Rodriguez-Boulan, Weill Cornell Medical College, New York), and ZO-1 (rat; MABT11; EMD Millipore). Rho activator II (CN03) was from Cytoskeleton (Denver, Colorado). Cycloheximide was from MP Biomedicals (Santa Ana, California) and AP21988 was from ARIAD Pharmaceuticals (Cambridge, Massachusetts). Phalloidin-CF405 and -Atto 647 N were purchased from Biotium (Fremont, California) and Sigma (St. Louis, Missouri), respectively. SNAP-Cell TMR-Star (TMR*) substrate was from New England BioLabs (Ipswich, Massachusetts).