Gel logic 2000

Total RNA was isolated from atrial and ventricular heart tissues using TRIzol reagent (15596-018; Invitrogen). cDNA was prepared from 1 μg of DNase treated RNA (4368813; Thermo Fisher Scientific). Primers targeting human DMPK exons 12–14 were used to assay transgene expression by real-time qPCR. Mouse Rpl4 was used as internal control for normalization. PCR reactions were carried out on an Applied Biosystems 7500 Fast Real-Time PCR System using PowerUp SYBR-Green PCR master mix (Thermo Fisher Scientific). Relative expression levels were determined by the 2^-ΔΔCt method. For analysis of alternative splicing events, primers annealing to flanking constitutive exons were designed. PCR products were resolved on a 5% polyacrylamide gel. Ethidium bromide stained RT-PCR bands were analyzed using Kodak Gel Logic 2000 and Carestream software. Percent spliced in (PSI) values were calculated using densitometry according to the equation: PSI = 100 X [Inclusion band/(Inclusion band + Skipping band)]. Primer sequences are provided in Supplemental Table 2 and Supplemental Table 3. See complete unedited blots in the supplemental material.

Gelatinase and casein zymography was performed using serum-free conditioned media from PC-3 Tet-On cells that were cultured for 48 hours with and without Dox before overnight incubation in serum-free media. Media were concentrated by centrifugation through Amicon-30 filters (Millipore). Equal amounts of total proteins were used to perform zymography assays as previously described [67 ]. Zymography gels were photographed on a Gel Logic 2000 imaging system with Carestream Molecular Imaging software (Kodak). Relative density of digested bands was determined using Image J (NIH, Bethesda, MD) software.