2 HDI improves cardiac output after toxicity. gel. Standards of uPLB Rabbit Polyclonal to SLC25A6 and S16-phosphorylated PLB (pPLB) were prepared by solid-phase peptide synthesis.20 Prestained, broad-range protein molecular weight SDS-PAGE standards (Bio-Rad), with molecular mass ranging from 7 to 205 kDa, were used as standards. The samples were electrophoresed at constant voltage (100 V) for 80 min. Western blot detection of phospholamban The proteins separated by electrophoresis were electrotransferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad), according to the method of Towbin et al.21 The western blot transfer was performed in the presence of Tris-glycine buffer (25 mM Tris, pH 8.3, and 192 mM glycine, containing 10% methanol) in a Transblot cell (Bio-Rad), at 280 mA constant current, for 50 min at 4C. The membranes were blocked with 2% nonfat dry milk, for 1 h, and then washed for 10 min, three times, with PBS, containing 0.1% Tween 20. The membranes were incubated with either of two primary antibodies, 1D11Ab or 285Ab, in blocking buffer. Anti-PLB monoclonal antibody 1D11 binds both phosphorylated and uPLB. Anti-phosphoserine PLB polyclonal antibody 285, which only binds PLB, phosphorylated Sennidin A at serine-16. Both were produced and purified as described previously.22 1D11Ab or 285Ab (7.2 mg/mL) was diluted between 1:2,000 and 1:3,000. After 1-h incubation, excess primary antibody was washed for 10 min, three times, with PBS, containing 0.1% Tween Sennidin A 20. The blots were subsequently incubated with secondary antibodies. 1D11 was incubated with 1 mg/mL stock solution of horseradish peroxidase-conjugated goat anti-mouse IgG (H+L)-HRP (Southern Biotechnology Associates, Inc., Birmingham, AL, USA), diluted between 1:1,000 and 1:2,000, in blocking buffer, without sodium azide, for 1 h at room temperature (RT). 285Ab was incubated with goat anti-rabbit IgG (H+L)-HRP (Sigma-Aldrich Corporation, St. Louis, MO, USA), diluted between 1:1,000 and 1:2,000, in blocking buffer, without sodium azide, for 1 h at RT. Excess secondary antibody was washed for 10 min, three times, with PBS, containing 0.1% Tween 20. The antigenCantibody complexes were visualized by staining for peroxidase activity with 3,3-diaminobenzidine (DAB) tablets (Sigma), as a substrate. The color reaction was stopped by washing with deionized water. The immunoblots were scanned by a densitometer, using the reflectance mode, and the bands were quantitated using the volume (area density) analysis method. Results Validation of methodology We first performed control experiments to demonstrate that we can detect uPLB and pPLB in porcine cardiac tissue. Synthetic uPLB and pPLB were used as standards (first six lanes of Fig. 1). 285Ab only detects pPLB (Fig. 1, top), whereas 1D11Ab detects both uPLB and pPLB, with a slight preference for uPLB (Fig. 1, bottom). Both antibodies have approximately linear sensitivity in the range of 6C25 ng of PLB. Thus, 285Ab and 1D11Ab provide accurate measures of pPLB content and uPLB content, respectively. Our ability to detect both forms of PLB in porcine cardiac tissue is illustrated in the right two lanes of Fig. 1, which represent samples taken from the right ventricles of control pigs. For the pig that was given no medications (?), negligible pPLB, equal to or below the background, was detected (Fig. 1, top), but the total PLB was substantial (Fig. 1, bottom, 17.50 ng PLB/ em /em g). Thus, less than 1% of PLB was phosphorylated for the pig receiving no medications. As a positive control, another pig was given isoproterenol, which is known to induce phosphorylation of PLB via the em /em -adrenergic receptor, with downstream signaling through protein kinase A (PKA).23,24 The pig received isoproterenol 5 (g/min for 2 h, resulting in the HR increasing from 90 to 175/ min. The pig Sennidin A was killed, and the cardiac tissues were harvested and analyzed, as described in Methods. Sennidin A Isoproterenol had no significant effect on the total amount of PLB in the right ventricle (Fig. 1, bottom right, +), but it did Sennidin A produce a significant level of pPLB (Fig. 1, top right, +), corresponding to 1 1.1 ng/ em /em g total protein, showing that.