Fig. 4. SCF^FBXL6 ubiquitinates p53 at K291/292.

a HEK293 cells were transfected with Flag-FBXL6 for 48 h and then treated with MG132 or DMSO for 5 h before lysis. Protein levels were analyzed by IB, with β-actin as a loading control. b Flag-FBXL6 WT or deletion mutants and HA-p53 were co-expressed in HEK293 cells for 48 h. Cells were harvested for IB analysis, with β-actin as a loading control (left). The intensity of HA-p53 expression for each group was quantified by densitometry, with β-actin as a normalizer (right). c In vivo polyubiquitination of p53 in the presence of FBXL6 or its deletion mutants. HEK293 cells were transfected with the indicated plasmids for 48 h, followed by MG132 treatment for 5 h before lysis. Immunoprecipitation of ubiquitin-conjugated p53 proteins was performed with anti-HA magnetic beads. Immuno-complexes and lysates (input) were analyzed by IB using the indicated antibodies. d In vivo polyubiquitination of p53 in the presence of p53, FBXL6 and WT Ub or its mutants. 48 h after transfection, HEK293 cells were treated with MG132 for 5 h and lysed in RIPA buffer. Immunoprecipitation and IB analysis were performed as described in (c). e Amino acid sequences of p53 from the indicated species are highly conserved around the K291/292 ubiquitination sites. f In vivo polyubiquitination of p53 and its mutants. HEK293 cells were transfected with Flag-FBXL6, Myc-Ub, and HA-p53 or its mutants as indicated. Immunoprecipitation and IB analysis were performed as described in (c). Data are shown as the means ± SD. *p < 0.05, **p < 0.01.