Transferrin alexa 594

Adult rat cardiomyocytes were plated on laminin. The medium was replaced with serum-free medium containing either 0.1% DMSO (vehicle control) or 30 μM Dyngo-4a (Selleckchem, MA) and incubated in a 37 °C tissue-culture incubator for 30 minutes. Recombinant human chymase (2.5 μg/ml, Sigma-Aldrich, MO) or transferrin-Alexa 594 (5 μg/ml, Life Technologies, OR) was added to the medium and incubated for an additional 2 hours at 37 °C. The cells were chilled on ice, and washed three times with PBS, then PBS + 0.5M NaCl, and 3 final PBS washes, and then fixed with 4% paraformaldehyde in PBS for 30 minutes. The uptake of Recombinant human chymase was identified with the human chymase antibody (1:50, Abcam ab2377) with the appropriate secondary antibody Alexa Fluor-594 (1:700, Invitrogen). A separate cohort were lysed in RIPA buffer containing Halt protease inhibitor cocktail (PIERCE), and separated on a Novex™ 6% Tris-glycine gel (Invitrogen) under reducing conditions. Proteins were transferred to a PVDF membrane and probed with myosin heavy chain antibody (1:100, DSHB MF-20) and HRP conjugated secondary antibody (GE Healthcare) then developed with Supersignal West Dura Substrate (PIERCE). Equal loading was confirmed by colloidal blue staining (Invitrogen).