Relative changes in the expression of a specific protein are commonly measured on western blots by forming the ratio of the densitometric values of bands containing the protein between control and experimental samples. It is generally assumed that this analysis provides an accurate determination of relative changes in a specific protein expression if there is a linear relation between increasing amounts of that protein, as represented by bands on a western blot or a gel, and the densitometric measurements of these bands. In this study, we provide direct evidence that this assumption is invalid because even in the presence of a linear relation, densitometric ratios differ substantially from know actual ratios of protein amounts. We present also the mathematical basis for this bias, and demonstrate that it can be circumvented by using a "ratio standard curve". Alternatively, the use of purified protein standards to plot a standard densitometry curve can solve this problem, but a survey of the literature shows that most investigators who use western blots to quantify relative changes in protein expression samples do not have standards for the specific proteins that they are investigating.To examine whether densitometric ratios accurately reflect ratios between protein amounts, we performed experiments to silence lamin C (a nuclear envelope protein) in U-373 MG glioblastoma cells. These cells were grown as described earlier [1], and were transfected with a lamin C specific siRNA (5′-CUGGACUUCCAGAAGAdTdT-3′ and 5′-UGUUCUUCUGGAAGUCCAGdTdT-3′ for the sense and anti-sense sequences, respectively) using siLentFect lipid reagent (Bio-Rad) according to the manufacturer's instructions. Control cells were treated with transfection reagent and vehicle. One and two days after transfection, the cells were processed for SDS-PAGE as described earlier [1]. Protein concentrations were measured with the bicinchoninic acid assay [2]. Gels were loaded with incremental amounts (4, 8, 20, 40, and 80 g) of total cellular proteins from control samples, as well as with 80 g of total cellular protein for experimental samples. Each loading was performed in duplicate, and all the loadings were applied to the same gel to insure identical treatment during the western blotting procedure, which was performed following standard methods [3]. In addition, to avoid band distortion, sample loadings were brought to the same final volume with sample buffer. After blocking in PBS plus 5% dry milk, the blots were incubated with a rabbit anti-lamin antibody (Chemicon, Temecula, CA) diluted at 1:100 in blocking solution, and then with goat anti-rabbit IgGs conjugated to peroxidase (KPL, Gaithersburg, MD) diluted at 1:1000 in blocking solution. After incubation of the blots Address correspondance to: Dr.

Pyridostigmine (PYR) is a carbamate cholinesterase (ChE) inhibitor used during the Persian Gulf War as a pretreatment against possible chemical nerve agent attack. Because of its quaternary structure, PYR entry into the central nervous system is limited by the blood-brain barrier (BBB). Following reports of unexplained illnesses among Gulf War veterans, however, central nervous system effects of PYR have been postulated through either stress-induced alteration of BBB permeability or via interactions with other neurotoxic agents. We evaluated the effects of daily physical (treadmill running) stress or daily exposure to a subclinical dosage of the organophosphate ChE inhibitor paraoxon (PO) on ChE inhibition in blood, diaphragm and selected brain regions in young adult male Sprague-Dawley rats following subacute PYR exposures. In physical stress studies, rats were placed on a treadmill for 90 min each day for 14 days just prior to PYR (0, 3, or 10 mg/kg per day) administration. In PO-PYR interaction studies, rats were treated with PO (0, 0.05, or 0.1 mg/kg per day) 1 h prior to daily PYR (0 or 3 mg/kg per day) administration for 14 consecutive days. Rats were evaluated daily for signs of cholinergic toxicity and were killed 1 h after the final PYR treatment. Forced running increased plasma corticosterone levels throughout the experiment (on days 1, 3, 7 and 14) when measured immediately after termination of stress. PYR-treated rats in the high dosage (10 mg/kg per day) group exhibited slight signs of toxicity (involuntary movements) for the first 6 days, after which tolerance developed. Interestingly, signs of cholinergic toxicity following PYR were slightly but significantly increased in rats forced to run on the treadmill prior to dosing. ChE activities in whole blood and diaphragm were significantly reduced 1 h after the final PYR challenge, and ChE inhibition in diaphragm was significantly greater in stressed rats than in non-stressed controls following high dose PYR (10 mg/kg per day). No significant effects of treadmill running on PYR-induced ChE inhibition in brain regions were noted, however. Repeated subclinical PO exposure had no apparent effect on functional signs of PYR toxicity. As with repeated treadmill running, whole blood and diaphragm ChE activities were significantly reduced 1 h after the final PYR administration, and ChE inhibition was significantly greater with combined PO and PYR exposures. Brain regional ChE activity was significantly inhibited after daily PO exposure, but no increased inhibition was noted following combined PO and PYR dosing. We conclude that, while some stressors may under some conditions affect functional signs of toxicity following repeated pyridostigmine exposures, these changes are likely to occur via alteration of peripheral cholinergic mechanisms and not through enhanced entry of pyridostigmine into the brain.

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