Figure 4

Numb nuclear localization regulates p53 stabilization. (a) Cytosolic and nuclear fractions of SCB29 and SCIET27 cells were revealed by western blot with anti-p53, anti-p21 and anti-numb antibodies. (b and c, left panels) Cytosolic and nuclear fractions of (b) SCIET27 cells and (c) pTα^−/− thymocytes treated with PMA at different times were revealed by western blot with anti-p53, anti-p21 and anti-numb antibodies. (Right panels) Total lysates of (b) SCIET27 cells and (c) thymocytes derived from pTα^−/− treated with PMA were revealed in western blot with anti-numb, anti-tubulin or anti-β-actin antibodies. (d, f and h) Whole-cell lysates of HEK293 cells transfected with Flag-p53 and Mdm2, (d) with or without Flag-Numb p66, (f) with or without Flag-Numb ΔN mutant or (h) with Flag-Numb p66 in the presence or absence of CA-PKCθ, were immunoprecipitated using an antibody against Mdm2 and were revealed in western blot with anti-flag (p53) and anti-Mdm2 antibodies. (Lower panels) Western blot of Numb and p53 on whole-cell extracts from (d, f and h). (e, g, i and j) Whole-cell lysates of HEK293 cells transfected with Flag-p53, Mdm2 and HA-ubiquitin, (e) with or without Flag-Numb p66, (g) with or without Flag-Numb ΔN mutant, (i) with Flag-Numb p66 in the presence or absence of CA-PKCθ or (j) with or without Myc-PTB Numb, and treated with 30 μM MG132 for 4 h, were immunoprecipitated using an anti-p53 antibody and revealed in western blot with anti-HA (against HA-flagged ubiquitin) and anti-p53 antibodies. All samples were supplemented with empty vector so that the final concentration of DNA was the same in all reactions. C, cytosolic fraction; N, nuclear fraction. Anti-tubulin and anti-laminB antibodies were used as markers of the cytosol and nucleus, respectively. All data are representative of three independent experiments