Immunohistochemistry
The C2C12 cells were reverse transfected with lipofectamine:DNA complexes prepared according to the manufacturer’s protocol (ThermoFisher, lipofectamine 3000).  The C2C12 cells were trypsinized, resuspended in media (DMEM+10%FBS, without antibiotics). The single cell suspension was added to the lipofectamine mix at a concentration of 105 cells/well of 6-well dish. The cells were plated on a 6-well plate and allowed to adhere overnight. Next day, the media was changed to DMEM+10%FBS, with antibiotics. The cells were treated with either DMSO or isoproterenol at the concentration of 1µM for 8 minutes. The cells were washed with 1XPBS, and fixed with 4% formaldehyde overnight at 4°C. The cells were permeabilized with 1XPBS-0.1%Trition, 3X, 5’ each, followed by blocking in 1XPBS-0.1%Trition+10%FBS for 1 hour and overnight incubation in primary antibody at 3 µgm/ml (Pde4D, catalog# PA521590 , Lot #VH3049391A),  followed by washes in 1XPBS-0.1%Trition, 3X, 5 minutes each, and overnight incubation in secondary antibody with conjugated Alexa-488. The cells were washed again in 1XPBS-0.1%Trition, 3X, 5 m minute each and imaged with SP8 confocal microscope.  The immunostaining for p-Desmin for done as described above except for p-Desmin primary antibody used at a concentration of 200 µgm /ml (p-Desmin Ser60, ThermoFisher, Catalog# PA5-38837).