Western blotting was performed as previously described (18 (link)). Proteins from homogenized left ventricles were separated by
10% or 15% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes, which
were incubated with mouse monoclonal antibodies for SERCA-2a (1:1000, Affinity
BioReagents, USA), phospholamban (PLB; 0.5 µg/mL, Affinity BioReagents), and PLB
phosphorylated at serine 16 (pPLBSer16 1:5000, Badrilla, UK). After they
were washed, the membranes were incubated with anti-mouse (1:5000, Stressgen, Canada)
or anti-rabbit (1:7000, Stressgen) immunoglobulin antibodies conjugated to
horseradish peroxidase. After they were thoroughly washed, immunocomplexes were
detected using an enhanced horseradish peroxidase/luminol chemiluminescence system
(ECL plus, Amersham International, UK) and film (Hyperfilm ECL International).
Signals on the immunoblot were quantified with the Image J computer program (NIH,
USA). Each membrane was reprobed to determine GAPDH expression using a monoclonal
mouse antibody (1:5000, Abcam Cambridge, USA).
10% or 15% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes, which
were incubated with mouse monoclonal antibodies for SERCA-2a (1:1000, Affinity
BioReagents, USA), phospholamban (PLB; 0.5 µg/mL, Affinity BioReagents), and PLB
phosphorylated at serine 16 (pPLBSer16 1:5000, Badrilla, UK). After they
were washed, the membranes were incubated with anti-mouse (1:5000, Stressgen, Canada)
or anti-rabbit (1:7000, Stressgen) immunoglobulin antibodies conjugated to
horseradish peroxidase. After they were thoroughly washed, immunocomplexes were
detected using an enhanced horseradish peroxidase/luminol chemiluminescence system
(ECL plus, Amersham International, UK) and film (Hyperfilm ECL International).
Signals on the immunoblot were quantified with the Image J computer program (NIH,
USA). Each membrane was reprobed to determine GAPDH expression using a monoclonal
mouse antibody (1:5000, Abcam Cambridge, USA).
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