Western Blotting
The C2C12 cells were transfected with mutant and control PDE4DIP plasmids (PDE4DIP-MUT and PDE4DIP-WT respectively) using lipofectamine and cultured in a 100 mm plates. At day 3, cells reached 70% confluency and plasmid expression was verified by percentage mCherry fluorescence (>50%) using confocal microscopy. Cells were stimulated with isoproterenol (1 uM) and harvested in protease and phosphatase inhibitor buffers at 8 minutes. Cells were quickly collected, placed in liquid nitrogen and subsequently prepared for western blot analysis. The western blot was carried out using standard procedures. Western blot analysis was performed to quantify phosphorylation changes in the beta-2-adrenergic receptor using the antibodies for p-beta adrenergic receptor Ser355, 356 (Catalog # PA538403, ThermoFisher), p-beta adrenergic receptor Ser346 (Catalog#Ab19281, Abcam), total beta-adrenergic receptor (Ab182136, Abcam) at a concentration of 1 µgm /ml.