Protein Expression Analysis in EAM Rat Myocardium

LV myocardium from male and female EAM rats was homogenized in Laemmli buffer (253mM Tris/HCL pH 6.8, 8% SDS, 40% glycerin, 200mM DTT, 0.4% bromophenol blue) (54 (link)). Proteins were quantified with the BCA Assay (Thermo Scientific Pierce Protein Biology, Germany). Equal amounts of total proteins were separated on SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. The membranes were immunoblotted overnight with the following primary antibodies: Col3A1 (1:400, Santa Cruz, USA), ERK (1:1000, Santa Cruz, USA), p-ERK (1:2000, Santa Cruz, USA), p38 (1:500, Santa Cruz, USA) and p-p38 (1:500, Santa Cruz, USA). Equal sample loading was confirmed through an analysis of actin (1:1500, Santa Cruz, USA). Immunoreactive proteins were detected with ECL Plus (GE Healthcare, UK) and quantified with ImageLab (Bio-Rad Laboratories, USA).