Journal: The Journal of Experimental Medicine
Article Title: The human Tp53 Arg72Pro polymorphism explains different functional prognosis in stroke
Figure Lengend Snippet: Arg 72 -p53 interacts directly with mitochondrial Bcl-xL and increases neuronal vulnerability to ischemia-induced apoptosis. (A) HEK293T cells were transfected with Arg 72 -p53 or Pro 72 -p53. Cytoplasmic (cyto) and mitochondrial (mito) fractions were isolated and analyzed by Western blotting. VDAC, control for the integrity of the mitochondria. PCNA, control indicating absence of nuclear contamination in the mitochondrial fractions. Black lines indicate that intervening lanes have been spliced out. (B) Rat cortical primary neurons were transfected with Arg 72 -p53-IRES-EGFP or Pro 72 -p53-IRES-EGFP. p53 colocalization with Bcl-xL was analyzed by immunofluorescence. DAPI, nuclear staining. (C) Rat cortical primary neurons were transfected with Arg 72 -p53-IRES-EGFP, Pro 72 -p53-IRES-EGFP, or Arg 72 -p53-IRES-EGFP + Bcl-xL. Apoptosis was measured 14 h later. (D) Neurons were transfected with empty vector (control) or the minimum amount of Arg 72 -p53-IRES-EGFP or Arg 72 -p53-IRES-EGFP cDNA not altering neuronal survival (0.08 µg/10 6 neurons) and were exposed to 100 µM glutamate for 5 min and incubated in culture medium for a further 8 h. Apoptosis was measured. (E) Neurons were transfected as described in D and were exposed to oxygen and glucose deprivation (OGD) for 1 h. Apoptosis was measured. (F) Neurons from Tp53 −/− mice were transfected with human BAC containing the entire Tp53 gene locus encoding either proline (p53 −/− Pro 72 ) or arginine (p53 −/− Arg 72 ) and were exposed to oxygen and glucose deprivation (OGD) for 3 h. Apoptosis was measured. The data in A and B represent four independent experiments. The data in C–F are means ± SEM of four different cell cultures. *, P
Article Snippet: Aliquots of cell, mitochondrial, or cytosolic extracts were subjected to SDS polyacrylamide gel (Mini-PROTEAN; Bio-Rad Laboratories) and blotted with anti-p53, anti-p21, anticleaved PARP-1 (BD), anti-Bax, anti–active caspase 3 (Santa Cruz Biotechnology, Inc.), anti-PUMA, anti-PERP, anti-AIP1 (Abcam), anti–cytochrome c (Santa Cruz Biotechnology, Inc.), anti-VDAC (EMD), anti-PCNA (BD), or anti-GAPDH (Sigma-Aldrich) overnight at 4°C.
Techniques: Transfection, Isolation, Western Blot, Immunofluorescence, Staining, Plasmid Preparation, Incubation, Mouse Assay, BAC Assay