Further studies to investigate precise mechanisms of action are warranted. Materials and Methods Reagents and antibodies PMA and PKC inhibitors (calphostin C, chelerythrine chloride, Ro-32-0432, safingol, http://www.selleckchem.com/products/pacritinib-sb1518.html rottlerin and a myristoylated pseudosubstrate PKC�� inhibitor) were purchased from Calbiochem (San Diego, CA). PMA and the PKC inhibitors were prepared as 10 mM stock solutions in dimethyl sulfoxide or distilled water. Rabbit anti-BART antibody (10090-2-AP) was purchased from ProteinTech (Chicago, IL). Monoclonal antibodies against ANX7 (610669), Rac1 (610650) and ��-catenin (610154) were obtained from BD Transduction Laboratory (Palo Alto, CA). Polyclonal antibodies against pan-PKC (sc-10800), PKC�� (sc-208), PKC��1 (sc-209), PKC��2 (sc-210), PKC�� (sc-213), PKC�� (sc-214) and PKC�� (sc-215) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
The rabbit anti-phospho-pan-PKC antibody (9379) was purchased from Cell Signaling (Grand Island, NY), and an anti-phospho-PKC�� antibody (ab23513) was obtained from Abcam (Cambridge, MA). Polyclonal antibodies against phospho-PKC��1 (sc-101776), -PKC�� (sc-101777), and -PKC�� (sc-12355) were purchased from Santa Cruz Biotechnology, and polyclonal antibodies against phospho-PKC��2 (07-873) and -PKC�� (07-877) were obtained from Millipore (Billerica, MA). Cell culture The human PDAC cell line S2-013, a subline of SUIT-2, was obtained from Dr. T. Iwamura [20]. The human PDAC cell line PANC-1 was obtained from ATCC. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS) at 37��C in a 5% CO2, humid atmosphere.
Immunoprecipitation and mass spectrometric analysis of BART S2-013 cells were lysed in lysis buffer [20 mM HEPES (pH 7.4), 100 mM KCl, 5 mM MgCl2, 0.5% Triton X-100, and protease inhibitor cocktail tablets (Roche, Penzberg, Germany)]. Equal amounts of S2-013 cell lysates were incubated with 2 ��g of anti-BART antibody or normal rabbit IgG (isotype control) and protein G Sepharose. Co-immunoprecipitated proteins were separated on a 4% to 20% gradient SDS-PAGE and then silver stained. Bands precipitated by the anti-BART antibody were excised, digested with trypsin and analyzed using a Q-TOF Ultima tandem mass spectrometer (Waters, Milford, MA) with electrospray ionization.
Database searches of the acquired MS/MS spectra were performed using MASCOT v1.9.0 (Matrix Science, Boston, MA). In vivo binding of BART with ANX7 S2-013 cells were lysed with lysis buffer and immunoprecipitated Entinostat with 2 ��g of anti-BART or anti-ANX7 antibody. To examine the interaction of endogenous BART with ANX7, immune complexes were analyzed by Western blotting with anti-BART and anti-ANX7 antibodies. Confocal immunofluorescence microscopy Cells were fixed with 4% paraformaldehyde, permeabilized with 0.