Fig 1

Wild-type p53, but not the mutant p53^R175H, is a substrate of CAPN3.

(A) Left panel: the CAPN3 recognition motif. Right panel: upper panel shows the simplified hu-p53 protein with five frequent mutated sites in the DNA-binding domain (DBD) highlighted. Lower panels depict the two putative CAPN3 recognition regions in p53. Numbers denote the position of amino acids in p53. ATG, start codon; TGA, stop codon. (B–D) In vitro assay of p53 degradation by CAPN3 or CAPN3^C129S. CAPN3, CAPN3^C129S, Myc-hu-Def, Myc-p53, and its mutant derivatives were expressed in 293T cells, respectively. Protein extracts were mixed as indicated and the mixture was incubated in the reaction buffer containing 5 mM CaCl₂ at 37°C for 0, 10, 30 min (B), or 30 min (C), or 45 min (D). GAPDH: loading control. Immunodetection of p53 was achieved by using a combination of monoclonal antibodies PAb240 and PAb1620 (B, D), or Anti-Myc tag antibody 9E10 (C). GAPDH was recognised by mouse monoclonal antibody 5-E10 (B) or rabbit monoclonal antibody EPR1977Y (C, D). (E) Left panel: diagram showing the CAPN3-pFastBac plasmid (baculovirus expression system) for transfecting the SF9 cells to express His-CAPN3. Right panel: Coomassie brilliant blue (CBB) staining picture showing total protein crude extract (total crude) and purified His-CAPN3 using the Ni-NTA agarose beads (purified). (F) In vitro assay of the degradation of Myc-p53^WT-HA, Myc-p53^R248W-HA, or Myc-p53^R175H-HA by the purified His-CAPN3 in the presence of 10 mM CaCl₂ or 20 mM EDTA as indicated at 37°C for 45 min. GAPDH: loading control. p53 was detected by a combination of monoclonal antibodies PAb240 and Pab1620. GAPDH was recognised by mouse monoclonal antibody 5-E10.