Fig. 1: CKS1 and p27 expression in neuroblastoma cell lines.
a Protein extracts from neuroblastoma cell lines (MYCN amplified = Kelly, IMR32, LAN5, LU-NB-1, LU-NB-2; non-MYCN amplified = SKNAS, SHEP, hNB), normal human dermal fibroblasts (hDF) or immortalised, non-tumourigenic, human fibroblasts (BJ) were subjected to western blot analysis with the indicated antibodies. b The selected neuroblastoma cell lines were cultured in the presence of increasing concentrations of Prozac and subjected to western blot analysis with a p27 antibody. Folds of p27 inductions relative to actin are indicated between the blots. Cells were lysed in RIPA Buffer (50 mM Tris-HCl, 1% NP40, 0.1% SDS, 150 mM NaCl) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Roche) for 30 min in ice. Insoluble material was removed by centrifugation (13,000 rpm for 20 min at 4 °C) and protein concentration was assessed by the method of Bradford. Equal amounts of protein were separated by SDS/PAGE on 15% polyacrylamide gel and transferred into nitrocellulose membrane. Membranes were blocked with 5% non-fat dry milk in PBS 0.1% Tween 20 for 1 h at room temperature and incubated with primary antibodies. The antibodies used were: N-Myc (sc-53993, Santa Cruz Biotechnology, 1:500 dilution), CKS1 (36-6800, Invitrogen 1:400 dilution), β-Actin (A5441, Sigma-Aldrich 1:40000 dilution), p27Kip1 (sc-1641, Santa Cruz Biotechnology 1:200 dilution). After washes, membranes were hybridised with appropriate horseradish peroxidase-conjugated secondary antibodies (rabbit and mouse). Detection was performed with Plus-ECL chemiluminescence kit (Bio-Rad, Hercules, CA, USA).
