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.