Journal:

Article Title: Peripheral Nerve and Brain Differ in Their Capacity to Resolve N,N-Diethyldithiocarbamate-mediated Elevations in Copper and Oxidative Injury

doi: 10.1016/j.tox.2010.04.018

Figure Lengend Snippet: GST-α, HO-1 and SOD1 protein expression in sciatic nerve and brain. (A) Representative western blot showing relative amounts of GST-α in proteins isolated from the brains of animals at the 8 week DEDC exposure time point as compared to controls. (B) Levels of GST-α in brain (open circles) and sciatic nerve (closed circles) determined by western blot. Optical density of GST-α was normalized to that of actin within the same sample and then to the mean value obtained for control samples on the same membrane (n =4 for controls and n=6 for exposed at 8 weeks; n=6 for controls and n=4 for exposed at all other time points). (C) Levels of HO-1 in brain (open circles) and sciatic nerve (closed circles) determined by western blot. Optical density determinations and exposure groups were identical to those used in (B) for GST-α. (D) Levels of SOD1 in brain (open circles) and sciatic nerve (closed circles) determined by western blot. Optical density determinations and exposure groups were identical to those used in (B) for GST-α. Error bars represent SE. ** p < 0.01 and * p < 0.05 relative to controls run on the same membrane by one-way unpaired students t-test.

Article Snippet: Nonspecific binding sites were blocked in blocking buffer (5% nonfat powdered skim milk in tris buffered saline, pH 7.4, containing 0.1% Tween-20 (TBST) at room temperature for 1 h. Membranes were then incubated with primary antibodies against either glutathione transferase-α (GST α) (GSTA11: rabbit anti-GSTα, Alpha Diagnostic, San Antonio, TX, dilution 1:2,000), heme oxygenase 1(HO-1) (OSA-111; mouse anti-HO-1, Stressgen, Ann Arbor, MI, dilution 1:2,500), Cu-Zn superoxide dismutase-1 (SOD1) (FL-154: sc-11407, Santa Cruz Biotechnology, Santa Cruz, CA, dilution 1:5,000), copper chaperone for SOD1 (CCS) (FL-274: sc-20141, Santa Cruz Biotechnology, Santa Cruz, CA, dilution 1:3,000) or prion protein (clone 8H4, Sigma, St. Louis, MO, dilution 1:5,000) overnight at 4 °C.

Techniques: Expressing, Western Blot, Isolation, Control, Membrane