Anti c myc antibody

Human breast cancer (MCF7) cells (cell density 3 × 10⁶) were seeded in 10-cm plates and transfected with c-myc plasmids. The cells were fixed with 1% formaldehyde at room temperature after 48 hrs post transfection and neutralized with glycine. The cells were collected, resuspended in CHIP lysis buffer and sonicated (Vibra-Cell™, Newtown, USA). Samples were incubated with protein-G beads that had been pre-incubated with 4-10 μg of anti-cmyc antibody (Gene tex Inc, San Antonio, Texas, USA) or negative control IgG (Sigma-Aldrich Co, St. Louis, MO, USA). Immunoprecipitates were washed by using washing buffer and reverse-cross-linked. Then the DNA was purified by using PCR purification kit purchased from MACHEREY-NAGEL, Duren, Germany. Finally, the purity of the DNA was quantified by using NanoDrop, performed PCR and data analyzed.

The standard procedure of Western blotting was performed as described previously (Lee et al., 2011 (link)). The membranes were probed with monoclonal antibodies specific for HCV NS5B (1:5000; Abcam, Cambridge, MA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000; GeneTex, Irvine, CA, USA), anti-COX-2 antibody (1:1000; Cayman, MI, USA), anti-MAPK (phosphorylated and unphosphorylated forms of ERK1/2, p38, and JNK), anti-IKKα, anti-phospho-IKKα/β (Ser176/180), anti-NF-κB, anti-IκB-α, anti-phospho-IκB-α (Ser32) (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), or anti-C-Myc antibody (1:1000; GeneTex, Irvine, CA, USA). The ECL detection kit was used for the signal detection (PerkinElmer, Shelton, CT, USA).

Western blotting was carried out as described previously [27 (link)]. In brief, an equal volume of cellular lysate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were transferred to a PVDF membrane. The membranes were probed with antibodies specific to DENV NS2B (1:5000; Abcam, Cambridge, MA, USA), anti-GAPDH (1:10,000; GeneTex, Irvine, CA, USA), anti-COX-2 antibody (1:1000; Cayman Chemical, Ann Arbor, MI, USA), anti-MAPK (phosphorylated and unphosphorylated forms of ERK1/2, p38, and JNK), and anti-C-Myc antibody (1:1000; GeneTex).