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Immunoprecipitation of glutathionylated proteins was carried out with a mouse monoclonal anti-GSH antibody at a concentration of 5 µg antibody/200 µg total protein (101-A, clone D8, Virogen, USA). All α-subunits were immunodetected with the rabbit monoclonal sc-28800 diluted 1:1000 (H-300, Santa Cruz Biotech). The β1 isoform was immunodetected with a polyclonal rabbit antibody generously provided by Dr Pedersen, University of Copenhagen. The β2 isoform was detected with the rabbit polyclonal antibody 06-1711 diluted 1:2000 (Millipore, USA).

All samples of PNS were tested for the content of the prototypical plasma membrane marker, Na, K-ATPase (EC 3.6.1.3). This was made by resolution by SDS-PAGE (+DTT) in 10% gel and immunoblotting with antibody oriented against α-subunit of this enzyme (sc-28800, Santa Cruz) as described by Dlouha et al. [19 (link)].
The content of Na, K-ATPase was also determined as the number of specific [³H]ouabain binding sites at saturating concentration of this inhibitor [20 (link)]. PNS samples were diluted to the same protein concentration in control (─M10, ─M10/─M20) and morphine-treated (+M10, +M10/─M20) samples and incubated with 18 nM [³H]ouabain for 90 min at 30ºC in total volume of 0.2 ml of 5 mM NaHPO₄, 5 mM MgCl₂, 50 mM Tris-HCl, pH 7.6. The binding reaction was terminated by dilution with 5 ml of ice-cold buffer and filtration through Whatman GF/B filters. The filters were washed twice and radioactivity remaining on the filters was determined by liquid scintillation using Rotiszcint Eco Plus cocktail. Non-specific binding was determined in the presence of 10 μM unlabelled ouabain.