Figure 2.
Stimulation of phospholamban phosphorylation on Ser-16 by depletion of Ca2+ from the SR of cardiac myocytes. The site-specific phosphorylation of phospholamban was measured by using antibodies specific to the phosphoprotein (9) following exposure of rat ventricular myocytes to Ca2+-pump inhibitors or the β-agonist, isoprenaline. Rat ventricular myocytes (1.1 × 105/ml, ref. 21) were exposed to 0.5% dimethylsulfoxide (control, Con), 2.5 μM Tg (TG), 30 μM t-BHQ (BHQ), 50 μM CPA, or 200 nM isoprenaline (Iso) for 5 min. Total cell protein (10,000 cells) was resolved by SDS/PAGE (13) and transferred to poly(vinylidene difluoride) membrane (9). The phosphorylation of phospholamban on Ser-16 was determined by using a phosphorylation site-specific antibody (9), developed by using an enhanced chemiluminescent substrate, and the region of the autoradiograph containing phospholamban is shown. Similar results have been obtained in six experiments with rat and ferret myocytes.