Mouse anti n myc antibody

For the analysis of protein expression by immunoblot, cells were lysed, protein extracted and separated by gel electrophoresis. After western transfer, membranes were probed with mouse anti-N-Myc antibody (1∶1000) (Santa Cruz Biotech), followed by horseradish peroxidase-conjugated anti-mouse (1∶10000) antiserum (Santa Cruz Biotech). Protein bands were visualized with SuperSignal (Pierce). The membranes were lastly re-probed with an anti-actin antibody (Sigma) as loading controls.

Gene expression in tumor cells was examined by quantitative real-time RT-PCR as described previously [21 (link), 36 (link)]. For the analysis of protein expression by immunoblot, cells were lysed, protein extracted and separated by gel electrophoresis. After western transfer, membranes were probed with mouse anti-N-Myc antibody (1:1000) (Santa Cruz Biotech, CA, USA) or rabbit anti-JMJD1A antibody (1:500) (Abcam, Cambridge, MA, USA), followed by horseradish peroxidase-conjugated anti-mouse (1:10000) or anti-rabbit (1:20000) antiserum (Santa Cruz Biotech). Protein bands were visualized with SuperSignal (Pierce, Rockford, IL, USA). The membranes were lastly re-probed with an anti-actin antibody (Sigma, St Louis, MO, USA) as loading controls.

Protein was extracted from tumor cells with radioimmunoprecipitation assay buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-Cl pH 7.5) containing protease inhibitors (Sigma, St Louis, MO, USA). Supernatant was collected after the samples were centrifugated at 13,000 × g at 4 °C for 20 min. Protein concentrations in the supernatant samples were quantified with the Bicinchoninic Acid Assay kit (Pierce, Rockford, IL).
For immunoblotting, protein samples were loaded onto sodium dodecyl sulfate-polyacrylamide gels, followed by electrophoresis and transfer to nitrocellulose membranes. The membranes were blocked of nonspecific antibody binding with 10% skim milk powder in phosphate-buffered saline, and probed with the following primary antibodies: mouse anti-E2F2 antibody (1:2000) (sc-633×), mouse anti-JMJD6 antibody (1:500) (sc-28348), mouse anti-N-Myc antibody (1:1000) (sc-53993), or rabbit anti-c-Myc antibody (1:500) (sc-764) (all from Santa Cruz Biotechnology). The membranes were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibody (1:10000) (both from Santa Cruz Biotechnology), and protein bands were visualized with SuperSignal (Pierce). The membranes were finally probed with a mouse anti-actin antibody (1:30000) (A3853, Sigma) as loading controls.