Dab nicl2

A sample of lysed ciliates prepared as previously described^{53 (link)}, treated under reducing conditions (after the addition of 200 mM of dithiothreitol-DTT) or under non-reducing conditions (without the addition of DTT), was separated by 12.5% linear sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using pre-stained molecular weight (MW) markers as molecular size standards^{53 (link)}. The separated proteins were electro-transferred to membranes of 0.45 µm PVDF (Hybond-P, GE Healthcare) in a semi-dry transfer system (Trans-Blot SD, Biorad), as previously described^{54 (link)}. The membranes were immunoblotted with a 1: 100 dilution of the anti-rPdVP1 (α-rPdVP1) and anti-KLH-HK (α-KLH-HK) antibodies and then with a polyclonal peroxidase-conjugated rabbit anti-mouse antibody (Dakopatts, Denmark) at 1: 800 dilution. The blots were stained by adding a chromogenic enzyme substrate solution consisting of 0.003% H₂O₂ and 0.06% 3,30-diaminobenzidine tetrahydrochloride with 0.03% NiCl₂ (DAB/NiCl₂, Sigma, USA)^{51 (link)}.

SDS-PAGE of the rAOX protein and a ciliate lysate (CL)—obtained from cultures maintained under normoxic or hypoxic conditions^{44 (link)}—and treated under reducing conditions (after the addition of 0.02 M dithiothreitol-DTT-) or under non-reducing (without the addition of DTT), was performed on linear 12.5% polyacrylamide minigels in a Mini-Protean Tetra cell system (BioRad, USA), as previously described^{35 (link)}. Once electrophoresis was completed, the gels were stained with a solution of GelCode Blue Stain Reagent (Thermo Scientific) according to the manufacturer's instructions.
Samples separated by electrophoresis were also analyzed by Western blot, with a slightly modified version of a previously described protocol^{35 (link)}. The samples were first incubated with serum from mice immunized with AOX (anti-rAOX serum) and then with a polyclonal peroxidase-conjugated rabbit anti-mouse antibody (Dakopatts, Denmark) at 1:800 dilution. The blots were stained by adding a chromogenic enzyme substrate solution consisting of 0.003% H₂O₂ and 0.06% 3,30-diaminobenzidine tetrahydrochloride with 0.03% NiCl2 (DAB/NiCl2, Sigma, USA).