Skip to main content
. 2018 Mar 16;7:e31275. doi: 10.7554/eLife.31275

Figure 4. SPIN1 blocks uL18-MDM2 interaction by sequestering uL18 in the nucleolus.

(A) SPIN1 overexpression attenuates p53 activation induced by ectopic uL18. U2OS cells were co-transfected with plasmids encoding Flag-uL18 or Myc-SPIN1 for 36 hr and harvested for WB analysis with indicated antibodies. (B) Overexpression of SPIN1 disrupts the uL18-MDM2 binding. Lysates were prepared from HCT116p53-/- cells co-transfected with HA-MDM2, Flag-uL18, Myc-SPIN1 or the corresponding empty vectors for 48 hr and analyzed by immunoprecipitated with the anti-HA antibody. Immunoprecipitates and 5% of inputs were immunoblotted with the indicated antibodies. (C) Overexpression of SPIN1 fails to disrupt the uL5-MDM2 interaction. Lysates were prepared from HCT116p53-/- cells co-transfected with HA-MDM2, Flag-uL5 and Myc-SPIN1 for 48 hr and analyzed by immunoprecipitated with the anti-HA antibody. Immunoprecipitates and 5% of inputs were immunoblotted with the indicated antibodies. (LC: light chain). (D) SPIN1 and uL18 co-localize in the nucleolus. H1299 cells were transfected with GFP-SPIN1 and Flag-uL18 for 36 hr and then immunostained with the anti-Flag antibody (red), and counterstained with DAPI.

Figure 4.

Figure 4—figure supplement 1. SPIN1 does not bind to MDM2, and SPIN1 and uL18 co-localize in the nucleolus.

Figure 4—figure supplement 1.

(A) SPIN1 does not bind to MDM2. HCT116p53-/- cells were transfected with combination of plasmids encoding Myc-SPIN1 and HA-MDM2, followed by IP-WB analysis with indicated antibodies. (B) SPIN1 and uL18 co-localize in the nucleolus. HEK293 and H1299 SPIN1 stable cells were transfected with Flag-uL18 for 36 hr and then immunostained with anti-Myc (red) and anti-Flag antibody (green), and counterstained with DAPI.