Fig. 6

DCAF13 inhibits p53 protein stability via ubiquitination modification. (A) A549 cells were transfected with siDCAF13#1, #2, #3 for 48 h. Before harvest, cells were treated with MG132 (5 μM) for 6 h, cell lysates were subjected to western blotting with indicated antibodies. (B) A549 cells were transfected with siDCAF13#1 for 48 h., Before harvest, cells were treated with 10 mg/mL cycloheximide (CHX) for the indicated time points, and then the cell lysates were used for western blotting. (C) The ubiquitinase and substrate website predicts that p53 is the most potentially substrate specifically recognized by DCAF13 using UbiBrowser database. (D) Immunofluorescence assay was performed to observe the cellular localization of DCAF13 and p53 in A549 and SPC-A1 cells. Scale bar = 50 μm. (E) A549 or SPC-A1 whole cell lysates were immunoprecipitated with protein-A/G agarose beads (mock IgG) or anti-p53 antibody and western blotted with indicated antibodies. Levels of endogenous p53, DCAF13, CUL4A, DDB1 and RBX1 were determined by western blot analysis of cell extracts (input) with indicated antibodies. F-H. A549 or SPC-A1 cells were co-transfected with the indicated siRNA and plasmids for 48 h. Before harvest, cells were treated with MG132 (5 μM) for 6 h. Subsequently, cell lysates were subjected to IP assays with p53 antibody for 12 h and then with protein G beads for 4 h. The mixtures including were ubiquitinated p53 were analyzed by western blot analysis with indicated antibodies. Polyubiquitination level of p53 was detected with the anti-His antibody. I. A549 or SPC-A1 cells were co-transfected with the indicated siRNA and plasmids, including K0 (lysineless), K48 (only K48-linked Ub), and K63 (only K63-linked Ub) as indicated, the following procedure is as shown in Fig. 6F-H