Journal: The Journal of Biological Chemistry
Article Title: TAp73 Induction by Nitric Oxide
doi: 10.1074/jbc.M110.184879
Figure Lengend Snippet: Mitochondrial membrane potential is compromised by knocking down TAp73. A, cells were transfected with 500 nm concentrations of a TAp73 siRNA or a siRNA control (Ctrl). Mitochondrial membrane potential ΔΨm was measured by flow cytometry 48 h later using the JC-1 probe. Some samples were treated by 0.5 mm DETA-NO or 0.1 mm H2O2 during the last 24 h or the last 6 h, respectively. The uncoupling agent carbonyl cyanide p-chlorophenylhydrazone (CCCP) at 50 μm was used as a positive control. Flow cytometry diagrams show the green fluorescent signal of JC-1 monomers in apoptotic cells on the x axis versus the red fluorescent signal of JC-1 oligomers in intact cells on the y axis. B, JC-1 green (gray bars) and red (black bars) fluorescence is shown. siCtrl and siTAp73 refer to a siRNA control and a siRNA against TAp73 mRNA, respectively. Results are the mean ± S.D. of three determinations. C, red-to-green fluorescence ratio was determined from values shown in B. This ratio is proportional to ΔΨm. D, cells were either not transfected (white bars) or transfected with a p53 (gray bars) or a p73 (black bars) siRNA. One day later they were treated with 6 μg/ml paraquat or 0.1 mm H2O2 as indicated. ROS production was analyzed after 24 h by flow cytometry using the H2DCFDA fluorogen. The results shown are the mean ± S.E. of three determinations. p < 0.05 (*) and p < 0.01 (**), compared with untreated and non-transfected cells.
Article Snippet: Primary antibodies to p73 (IMG-246, Imgenex, and A300-126A, Bethyl Laboratories), Chk1, NQO1, and Crk-L (sc-8408, sc-32793, and sc-319 respectively, Santa Cruz Biotechnology), phospho-Chk1 and phospho-Chk2 (2348 and 2661 respectively, Cell Signaling), β-actin and α-tubulin (A 5316 and T 9026, Sigma) were used for immunoblotting.
Techniques: Membrane, Transfection, Control, Flow Cytometry, Positive Control, Fluorescence
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