Journal: Cell Death and Differentiation

Article Title: Post-translational regulation of p53 function through 20S proteasome-mediated cleavage

doi: 10.1038/cdd.2017.139

Figure Lengend Snippet: Cellular FLp53 post-translational processing by the 20S proteasome generates the Δ40p53 isoform. (a) The non-small-cell lung carcinoma cell line, H1299, which is p53 deficient, was transiently transfected with pC53-SN3 vector expressing FLp53, followed by cell lysis. Cells extracts were incubated with the indicated elevated concentrations of 20S proteasome for 120 min at 37 °C and p53 forms were detected by SDS-PAGE. (b) H1299 cells were transiently transfected with pC53-SN3 vector expressing FLp53 followed by cell lysis. Cells extracts were incubated with 20S proteasome (10 μg) in the presence or absence of the proteasome inhibitor MG132 (50 μM) for 120 min at 37 °C and p53 forms were detected by SDS-PAGE. (c) H1299 cells were transiently transfected with pC53-SN3 vector expressing FLp53. After 24 h, cells were treated with proteasome inhibitor MG132 (10 μM) for 6 h. Cells were lysed and cell extracts were loaded on SDS-PAGE gel. Cells expressing an empty vector (Con) were used as negative controls. (d) H1299 cells were transiently transfected with the following vectors: pC53-SN3 vector (FLp53), pC53-SN3 vector containing a single in-frame mutation, substituting methionine (ATG) in position 40 to isoleucine (ATA) (p53M40I) to eliminate translation initiation from the alternative site and an empty vector (Con). Cells were lysed and cells extracts were loaded on SDS-PAGE gel. (e) HEK293T cells were transiently transfected to silence Rpn2. As controls, non-targeting siRNA (NT) was used. After 48 h, cells were treated with proteasome inhibitor MG132 (40 μM) for 3 h. Cells were lysed and cell extracts were loaded on SDS-PAGE gels, analyzed by western blot and visualized using various antibodies. In all experiments, the polyclonal anti-p53 antibody was used to detect p53 forms. The housekeeping protein GAPDH was used as a loading control. Purified Δ40p53 was loaded on gels as a control. Blots are of a representative experiment. Quantification demonstrates the average of four (a, c–e) or five (b) independent experiments. Measurements were subjected to Student’s t-test analysis, *P⩽0.05, **P⩽0.01. Error bars represents S.E. ND, not detected

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p53, H47 (produced in our laboratory); goat polyclonal anti-p53 horseradish peroxidase-conjugated (R&D Systems, Minneapolis, MN, USA) PAb421 mouse monoclonal anti-p53 directed to the C-terminal were purified from ascetic fluids, mouse monoclonal anti-p53 antibody, DO-1, directed against residues 21–25 in p53 N′ terminus that are not included in the Δ40p53 isoform (kindly provided by Prof. Sir David Lane (p53 Laboratory, A*STAR, Singapore).

Techniques: Transfection, Plasmid Preparation, Expressing, Lysis, Incubation, SDS Page, Mutagenesis, Western Blot, Purification

Journal: Cell Death and Differentiation

Article Title: Post-translational regulation of p53 function through 20S proteasome-mediated cleavage

doi: 10.1038/cdd.2017.139

Figure Lengend Snippet: Hetero-tetrameric p53 is formed through the association of FLp53 and Δ40p53 dimers. FLp53 and Δ40p53 were mixed in a ratio of 1 : 1, in the absence (a) and presence (b) of DNA (p53r2 RE), followed by native MS analysis of the species generated after overnight incubation at 4 °C. (c) H1299 cells were transiently transfected with pC53-SN3 vector expressing FLp53 (FLp53) or with an empty vector as a negative control (Con). After 48 h, cells were lysed and cells extracts were incubated with either monoclonal antibody against p53 N-terminus (DO-1) that is directed to an epitope that does not appear in Δ40p53 protein, in order to immunoprecipitate only FLp53, or with a non-specific IgG antibody as a negative control. Immunoprecipitates were loaded on SDS-PAGE gel, and p53 forms were detected using goat polyclonal anti-p53 horseradish peroxidase-conjugated antibody (p53HRP). To examine p53 expression, non-immunoprecipitated samples were loaded (input). Purified Δ40p53 was loaded as a positive control. This figure combines two exposures of the same gel, which are separated by a vertical line; the long exposure of the immunoprecipitated samples is shown on the left and the short exposure of the highly concentrated purified Δ40p53 protein is displayed on the right. The two original images are presented in Supplementary Figure 3. (d) HEK293T cells were lysed and cells extracts were incubated with either DO-1 antibody, to immuneprecipitate FLp53, or with a non-specific IgG antibody as a negative control. Immunoprecipitates were loaded on SDS-PAGE gel, and p53 forms were detected using p53HRP antibody. To examine p53 expression, non-immunoprecipitated samples were loaded (input). Purified Δ40p53 was loaded as a positive control. Results are a representative experiment of at least three independent experiments

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p53, H47 (produced in our laboratory); goat polyclonal anti-p53 horseradish peroxidase-conjugated (R&D Systems, Minneapolis, MN, USA) PAb421 mouse monoclonal anti-p53 directed to the C-terminal were purified from ascetic fluids, mouse monoclonal anti-p53 antibody, DO-1, directed against residues 21–25 in p53 N′ terminus that are not included in the Δ40p53 isoform (kindly provided by Prof. Sir David Lane (p53 Laboratory, A*STAR, Singapore).

Techniques: Generated, Incubation, Transfection, Plasmid Preparation, Expressing, Negative Control, SDS Page, Immunoprecipitation, Purification, Positive Control