All reagents were purchased from Sigma (St. Louis, MO) except where indicated. Rp-cAMPS and Sp-cAMPS were purchased from BioLog (La Jolla, CA). Bisindolylmaleimide I (GF109203X) and Et-18-OCH3 were purchased from Calbiochem (San Diego, CA). 2, 10, 11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) was purchased from Research Biochemicals Inc. Protease inhibitor tablets (complete) were purchased from Roche Molecular Biochemicals (Indianapolis, IN).
NG108-15 cells stably expressing the rat D2L receptor (NG108-15/D2) (Asai et al., 1998
) were grown on single-well slides in defined media for 2 days followed by daily replacement until day 4 (Dohrman et al., 1996
). The cells were treated as described in the figure legends and fixed as described below (Gordon et al., 1997
Immunocytochemistry and Microscopy
Cells were fixed with cold methanol for 2 to 3 min and rinsed 3 times with PBS, incubated at room temperature with blocking buffer (1% normal goat serum in PBS and 0.1% Triton X-100) for 3 to 4 h, and then incubated overnight at 4°C in PBS containing 0.1% Triton X-100, 2 mg/ml fatty acid-free bovine serum albumin (Dohrman et al., 1996
), primary antibodies specific for εPKC (mouse IgG raised against the V5 domain of εPKC, Santa Cruz Biotechnology, Santa Cruz, CA), εRACK (rat IgG, Stressgen, Victoria, BC, Canada) for εRACK, and 14E6 (mouse IgM, raised against the V1 domain of εPKC) for active εPKC (Souroujon et al., 2004
). The cells were then washed three times with PBS, incubated for 1 h at room temperature with goat anti-mouse IgM, anti-mouse IgG, or anti-rat IgG secondary antibodies (Cappel, Aurora, OH) (diluted 1:1000), washed three times with PBS, and coverslipped with Vectashield mounting medium. Cells were imaged using a Bio-Rad 1024 scanning laser confocal microscope equipped with a krypton-argon laser attached to a Nikon Optiphot microscope. Images were collected as z-series using Kalman averaging of scans (Gordon et al., 1997
). Collected data were processed using NIH Image and Adobe Photoshop software (Adobe, Mountain View, CA). All images were obtained under 40x magnification from individual middle sections of the projected z-series.
Quantification of PKC Localization
Fields on each slide were selected at random and cells scored for perinuclear or cytoplasmic staining by two independent observers who were blind to the experimental conditions. At least four fields were scored for each experiment, for a total number of at least 50 cells per slide.
NG108-15/D2 cells in 100 mm dishes (2 × 106
cells/dish) were incubated with ethanol or NPA for 10 min, washed with cold PBS and lysed on ice in 0.5 ml lysis buffer containing 50 mM Tris-HCL (pH 7.4), 2.5 mM MgCL2
, 1 mM EDTA, 1 mM DTT, 10% glycerol and protease inhibitors (0.1 mM phenylmethyl sulfonyl fluoride, 20 μg/ml soybean trypsin inhibitor, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 1 mM sodium orthovanadate). Cells were homogenized by ten passes through a 26-gauge needle and centrifuged at 3000 rpm for 5 min at 4°C. The supernatant was centrifuged for 20 min at 150,000 × g to separate the membrane pellet from the cytosol (Yao et al., 2002
). The supernatant was saved as the cytosolic fraction. The remaining pellets were suspended in 0.5 ml of lysis buffer containing 0.1% Triton-X 100, titrated and incubated on ice for 20 min. This suspension was centrifuged as described above, and the Triton-soluble material was collected as the original particulate fraction.
Immunoprecipitation and Western Blot
5 μg of εPKC monoclonal IgG antibody was incubated with 50 μl of protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. Antibody-bound beads were then washed twice with PBS and blocked with 3% BSA for 2 h at 4 °C. The cytosolic fraction was precleared with protein A/G beads for 30 min at 4 °C, incubated with the antibody-bound beads overnight at 4 °C and subsequently washed four times with PBS. Bound material was eluted with SDS sample buffer, run on a 10% SDS/PAGE and transferred and probed for εPKC (mouse IgG, Santa Cruz, CA) and εRACK (rat IgG, Victoria, BC). Secondary antibody was horseradish peroxidase-linked goat anti-mouse or anti-rat (1:1000) (NEN BioLabs, Beverly, MA). Proteins were detected using LumiGLO chemiluminescence substrate (NEN BioLabs).
PKC Activity Assay
Cells grown in 100 mm plates were treated with ethanol or NPA for 10 min, washed with cold PBS, harvested in 1 ml whole cell lysis buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 10 mM EGTA, 0.1% Triton-X 100, and 1 tablet of protease inhibitor/10 ml), and lysed on ice for 20 min. The lysate was centrifuged at 14,000 rpm for 10 min in an Eppendorf centrifuge. The supernatant was immunoprecipitated for εPKC as described above. To assay εPKC activity, immunoprecipitates were incubated at 30 °C for 20 min with 10 μM ATP, 0.5 μci [γ-32P]ATP and a peptide substrate mixture from SignaTECT PKC Assay System (Promega, Madison, WI). PKC activity was detected as described by the manufacturer.