Mouse monoclonal anti hdac1

Western blotting was performed as previously described (14 (link)) with minor modifications. Cellular proteins were isolated using 1 × RIPA buffer containing a protease inhibitor and a phosphatase inhibitor. The proteins were resolved by electrophoresis in a 10–15% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (GE Healthcare). The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20. Rabbit monoclonal anti-STING (Cell Signaling Technology, Beverly, MA), Rabbit monoclonal anti-cGAS (Cell Signaling Technology), rabbit polyclonal anti-Rab27b (Bioss Antibodies Inc., Woburn. MA), mouse monoclonal anti-KSHV ORF65 (14 (link)), rabbit polyclonal anti-GAPDH (Cusabio, Houston, TX), rabbit polyclonal anti-calnexin (Bioss Antibodies Inc.), mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-mtTFA (Santa Cruz Biotechnology), rabbit polyclonal anti-MX1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT44L (Bioss Antibodies Inc.) and mouse monoclonal anti-β-actin antibodies (Sigma, St. Louis, MO) were used as primary antibodies. HRP-conjugated anti-rabbit or anti-mouse antibodies (Bethyl Laboratories Inc., Montgomery, TX) were used as secondary antibodies. The results were visualized using an ECL detection reagent (Bio-Rad, Hercules, CA).

NB cells were seeded on 12-mm glass coverslips (2.5 × 10⁵ cells) that were placed in a 24-well-plate. On the second day after seeding, cells were incubated in serum-free media for 24 h before stimulation with 10⁻⁸ M NPY for 20 min, with or without a 30-min pre-incubation with 10⁻⁶ M Y5R antagonist. The cells were fixed for 10 min using 4% paraformaldehyde followed by permeabilization for 10 min. To test the interaction between Y5R and RhoA-GTP, Duolink® In situ Red starter kit Mouse/Rabbit (Sigma-Aldrich) was used according to the manufacture's protocol with the following antibodies: rabbit monoclonal anti-Y5R (1:250; Novus Biologicals; cat # NBP1-00957), mouse monoclonal anti-RhoA-GTP (1:100; NewEast Biosciences; cat # NE-26904), and mouse monoclonal anti-HDAC1 as a negative control (1:50, Santa Cruz Biotechnology; cat# SC-8410). For actin filament detection, Alexa Fluor 488-Phalloidin (1:100; Molecular Probes) was used for 20 min before mounting. Intensities of fluorescent staining in the entire cell colonies, their outer plasma membranes, and inner areas were quantified using Image J software.