Gel logic 6000 pro imaging system

The following antibodies were used for immuno-analyses: anti-RND3 (Cocalico Biologicals), anti-Snail1 (Santa Cruz, sc-28199), anti-E-cadherin (CST, 3195S), anti-claudin (Bioword, BS1063), anti-c-myc (9E10, Santa Cruz, sc-40), and anti-HA (Santa Cruz, SC-7392, SC-805). Even protein loading in the immunoblotting analysis was verified by the intensity of the GAPDH blot (Santa Cruz, sc-20357). The immunostaining, immunoblotting, and immunoprecipitation were conducted as described previously [8 , 20 (link)]. The immunoblotting densitometry was quantified by the Gel Logic 6000 PRO Imaging System (Carestream Health, Inc. Rochester, NY, USA), and the immunofluorescent and immunohistochemical image quantifications were conducted by Leica Application Suite Imaging Software (Version 4.0, Biberach, Germany).

Cardiac MyHC isoforms were separated by discontinuous gel electrophoresis. Briefly, frozen samples of left ventricle were homogenized on ice in a solution containing 50 mM potassium phosphate buffer (pH 7.0), 300mM sucrose, 0.5mM dithiotreitol (DTT), 1mM ethylenediaminetetraacetic acid (EDTA), 0.3mM phenylmethylsulfonyl fluoride (PMSF), 10mM sodium fluoride and protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Homogenates were centrifuged at 12000g for 20min at 4°C and total protein quantification was performed in supernatant aliquots using BCA Protein Assay kit (ThermoScientific, Wilmington, DE, USA). Samples were diluted to a final concentration of 1μg of protein/μL in a solution containing 65% (vol/vol) glycerol, 2.5% (vol/vol) β-mercaptoethanol, 1.15% (wt/vol) SDS, and 0.45% (wt/vol) Tris-HCl (pH 6.8). Then, 12ug of total protein was loaded into SDS-PAGE vertical slab gels (Hoefer, Holliston, MA, USA). The gels were run for 40 h at 20°C and constant voltage of 100 V. Protein bands for alpha and beta MyHC isoforms were visualized with Coomassie blue. Gels were digitally photographed with the Gel Logic 6000 Pro Imaging System (Carestream Health, Rochester, NY, USA), and analyzed for relative MyHC isoform percentages by densitometry using Image J Analysis software.