yields an amplicon between 80 and 200bp, spans exon-exon junctions, and has a melting point from 59-

Transcription

1 Supplemental Materials and Methods RTqPCR. All primers are listed in Supplemental Table 1 and were designed ensuring that each primer set yields an amplicon between 80 and 200bp, spans exon-exon junctions, and has a melting point from C. Amplicon quality was ensured using mfold (1) to ensure the absence 3 and 5 hairpin structures. Amplification efficiencies were determined for each primer set using 4 orders of magnitude serial dilutions of cdna template. All primer sets exhibited amplification efficiencies between %. Small sections (~35mg) of heart ventricle isolated from whole heart samples were rinsed with PBS, and immediately flash frozen in liquid nitrogen. The sections were then cut and placed on dry ice. Tissue homogenization was performed using a cooled (< 10 C) Precellys 24 Dual ceramic bead homogenizer (Bertin) (5000rpm, 2x20 second cycle, mixed 2.8mm large and 1.4mm small and beads). Total RNA was isolated using the RNeasy Mini Kit (#74104, Qiagen) following the manufacturer s directions. RNA quality and quantity was assessed using a BioAnalyzer 2100 (Agilent). Only samples with a RIN > 7.0 were used for further analysis. Reverse transcription was performed using the Maxima First Strand cdna Synthesis Kit (#K1642, Thermo Scientific). RTqPCR was performed in a BioRad icycler machine using Maxima SYBR Green/Fluorescein qpcr kit (#K0242, Fermentas/Thermo Scientific). Each PCR reaction contained 25ng of cdna template and forward and reverse primers at a final concentration of 0.2uM. For each sample a reverse transcription negative (NoRT) control was included, and for each gene on every plate a no template control was run. The PCR amplification protocol was as follows: 10min at 95 C (dwell time and well factor collection), 10 min at 95 C (initial denaturation), followed by 40 cycles of 15 seconds at 95 C, 30 seconds at 60 C, and 30 seconds at 72 C. Following amplification, a melting curve analysis was performed from C in 0.5 C steps to determine specificity of amplification and to check for primer dimer formation. Subsequently, one sample from each triplicate was separated on a 1.5% agarose gel to check for a single PCR product band. Final analysis of gene expression levels was determined using qbase v2.4 with per gene efficiency correction and geometric normalization based on three reference genes picked from a panel of eight candidate genes based on a combined GeNorm M value < 0.15 and in compliance with MIQE guidelines (2). References

2 genes were as follows: heart (Hprt, Rpl38, Rps3), liver (Eef2, Rps3, Actb), kidney (B2m, Hprt, Eif3f), and brain (B2m, Eef2, Rpl38). Please see supplemental table 1 for specific primer details. Immunoblots. Protein was isolated from sections of flash frozen heart, liver, or white adipose tissue (~30mg) from ic-ghrko and littermate controls and homogenized in RIPA buffer. Protein concentration was determined using a Bradford assay (BioRad, Hercules, CA) following the manufacturers directions. Protein samples (20µg) were separated on 10% SDS-polyacrylamide gels before being transferred to PVDF membranes (#RPN2020LFP, GE Life Sciences, Buckinghamshire, UK). Subsequently, membranes were blocked in 5% fat free dry milk (Carnation, Nestle, Vevey, Switzerland) in 0.5% TBS/Tween-20 and probed overnight with primary antibodies at the corresponding dilutions: pan Akt (#4685, 1:1000), p-akt (Ser473) (#4058, 1:1000), p42/44 MAPK (#4695, 1:1000), and p-p42/44 MAPK (#4377, 1:1000) (Cell Signaling, Danvers, MA). Membranes were incubated with goatanti rabbit HPR conjugated secondary antibody (1:50,000) (GE Life Sciences, Buckinghamshire, UK) in 5% fat free dry milk / 0.5% TBS/Tween-20 for 1 hour at room temperature before being subsequently washed in TBS and covered with ECL Prime reagent (GE Life Sciences, Buckinghamshire, UK) for 60 seconds. Finally, membranes were exposed to CLExposure X-ray Film (Thermo Fisher, Waltham, MA) and developed using an automatic film developer. Densitometry analysis was performed using ImageJ software (3). The following primary antibodies were used for STAT5 (#SC-835, 1:200, Santa Cruz Biotechnology, Dallas, TX) and p-stat5 (#9351, 1:1000, Cell Signaling, Danvers, MA).

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4 Supplemental Figure 1. Systolic blood pressure. Non-invasive tail-cuff systolic blood pressure was measured from months of age in ic-ghrko (N=8) and Control (N=24) littermates. * significant difference between genotypes (P<0.05), significant difference with age (P<0.05). ic- GHRKO: inducible, cardiac-specific GHR gene disrupted mouse, SBP: systolic blood pressure.

5 Supplemental Figure 2: Cardiac calcium channel mrna levels. Expression of the major cardiac calcium SERCA2, NCX1, LTCC, and RYR2 in 4.5 month old (A) and 12.5 month old (B) ic-ghrko (N=5) and control (N=15) littermates. * Significant difference between genotypes (P<0.05). ic-ghrko: inducible, cardiac-specific GHR gene disrupted mouse, SERCA2: sarcoplasmic endoreticulum calcium exchanger 2, LTCC: L-type calcium channel, NCX1: sodium calcium exchanger 1, RYR2: ryanodine receptor 2. References 1. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res Jul 1;31(13): Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem Apr;55(4): Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods Jul;9(7):671 5.