Fig. 3. FBXL6 destabilizes p53.

a p53 was identified as a FBXL6-interacting protein. HEK293 cells expressing Flag-tagged FBXL6 and control vector were treated with MG132 for 5 h and the lysates were subjected to immunoprecipitation using anti-Flag affinity columns. The eluates were resolved by SDS-PAGE and Coomassie blue staining. The protein bands were excised and analyzed by mass spectrometry. b Interaction between endogenous FBXL6 and p53. RKO and HCT116 cells were treated with MG132 for 5 h before being collected. After lysis, the supernatents were subjected to immunoprecipitation using the antibodies against the indicated proteins. Immuno-complexes and lysates (input) were analyzed by IB analysis with the indicated antibodies. c, d Schematic diagram showed the structure of FBXL6 (left) and p53 (right) and their deletion mutants used. Flag-FBXL6 or its mutants were co-expressed with HA-tagged p53 in HEK293 cells for 48 h (c). Flag-p53 or its mutants were co-expressed with HA-FBXL6 in HEK293 cells for 48 h (d). Extracts were immuno-precipitated with anti-Flag beads and examined by IB with the indicated antibodies. e FBXL6 affects p53 protein turnover. RKO cells stably expressing the indicated plasmids were treated with CHX and collected at the indicated times for IB analysis. β-actin was used as a loading control. f The intensity of p53 expression for each time point in (e) was quantified by densitometry, with β-actin as a normalizer. g RKO cells stably expressing indicated plasmids were treated with 0.5 µM DOX and collected at the indicated times for IB analysis. β-actin was used as a loading control. S: Short exposure, L: long exposure. Data are shown as the means ± SD. *p < 0.05, **p < 0.01.