Fig. 11
NBPF1 overexpression increases p21CIP1/WAF1 protein levels in NB cell lines with mutant p53. (A) SH-SY5Y, NLF and SK-N-AS cells were transfected with plasmids encoding either EGFP-luciferase or EGFP-NBPF1. Both EGFP-positive (+) and EGFP-negative populations (−) were isolated by FACS, and the protein levels of p21CIP1/WAF1 were detected by western blotting (top row). Treatment of HEK293T cells with doxorubicin (+ Doxo) served as a positive control for p21 induction. Detection with the anti-NBPF antibody (sc-82241) showed successful isolation of EGFP-NBPF1 transfected cells (second row). Detection with an anti-EGFP antibody (third row) showed successful isolation of transfected cells that were EGFP-luciferase positive (indicated by *) or EGFP-NBPF1 positive (indicated by **). Actin expression acted as a loading control (bottom row). (B) Quantification of p21CIP1/WAF1 signals in the blot of (A), normalized against actin signals. In addition to the positive control (HEK293T + Doxo), only NB cells with mutant p53 expressing EGFP-NBPF1 showed clear induction of p21. p-values were calculated with one-way ANOVA