Fig. 2

Chronic oxycodone treatment increases protein carbonyl content in rat plasma. a Dot-blot analysis of carbonylated proteins in rat plasma. Left panel, equal amount of total protein from plasma of rats treated with oxycodone (O1, O2) or water (W) were derivatized with DNPH, spotted on NC membrane and probed with anti-DNP antibodies. The same membrane was later stained with Ponceau S to detect total protein in each spot. Each plasma sample corresponds to the same animal from which cortex samples was obtained (Fig. 1a, Exp. #). The corresponding experiment is indicated above each set of samples. Right graph, quantitative analysis of dot-blot images shown on left. DNP signal was normalized to Ponceau S signal in corresponding sample and then oxycodone value was normalized to water value in the same experiment. Graph represents mean value of DNP to Ponceau S ratio normalized to water samples set as one (± SEM; n = 3 (three sets of experiments that analyzed samples from 9 water and 13 oxycodone treated rats; p = 0.1). b Dot-blot analysis of Triton™ X-100 insoluble carbonylated proteins in rat plasma. Equal volume of plasma samples from rats treated with water (W) or oxycodone (O) corresponding to samples presented in a was centrifuged at 20,000×g for 30 min. The pellets were resuspended in buffer containing 1% Triton™ X-100 and centrifuged again. Resulting pellets were dissolved in buffer, derivatized with DNPH, spotted on NC membrane and probed with anti-DNP antibodies