Fig. 6: ZCCHC10 protein interacts and stabilizes p53 protein.

a Immunofluorescence staining in Beas-2b cells. The murine anti-p53 monoclonal antibody and Texas Red-conjugated anti-mouse IgG (red) were used to detect p53, whereas rabbit anti-ZCCHC10 polyclonal antibody and FITC-conjugated anti-rabbit IgG (green) were used to detect ZCCHC10, respectively. Nuclei were stained by Hoechst 33258 (blue). Yellow in merged image represents colocalization of p53 and ZCCHC10 protein. Bar length: 10 μm. b, c Co-immunoprecipitation (co-IP) assays were performed in Beas-2b cells (b) or the Hek293 cells (c) co-transfected with HA-p53 and Myc-Zh10 (or Myc-mtZh10). Cell lysates were precipitated with the indicated antibody, and the immune complexes were subjected to WB. Input is equivalent to 10% of the lysate used for the co-IP. d WB analysis of p53 and Myc-ZCCHC10 protein in Hek293 cells at 24 h after transiently transfected with an increasing amount of empty vector, Myc-Zh10, or Myc-mtZh10 plasmids. e WB analysis of endogenous p53 and ZCCHC10 in Beas-2b stably expressing scramble shRNA (shNC) or ZCCHC10 shRNA (shZh10) and A549 cells stably expressing vector (Vec) or ZCCHC10 (Zh10). f Ubiquitination status of p53 following overexpression or knockdown of ZCCHC10. Cells were treated with MG132 for 4 h. After treatment, cell lysates (800 μg) were prepared and IPed with an anti-p53 antibody. Then, the precipitated proteins were subjected to WB using an anti-ubiquitin antibody to detect the ubiquitinated p53 (Ub-p53) protein (upper panel). Input is equivalent to 10% of the lysate used for the IP (lower panel). g Co-IP assays were performed in Hek293 cells co-transfected with the indicated amount of HA-p53, Myc-MDM2C464A, and EGFP-ZCCHC10 plasmids. At 24 h post transfection, cell lysates were prepared and IPed with rabbit anti-HA antibody, and the precipitated proteins were detected by WB analysis using a mouse anti-HA, anti-Myc, and anti-EGFP antibodies. Input is equivalent to 10% of the lysate used for the IP