Typhoon 9400

After SDS-PAGE, the gels were scanned with a Typhoon 9400 fluorescence gel scanner (GE Healthcare, Piscataway, NJ) using appropriate individual excitation and emission wavelengths, filters and photomultiplier (PTM) values that are sensitive for each of the Cy3, Cy5 and Cy2 dyes (PTM values: 480 nm, 490 nm, 500 nm, respectively). Relative protein quantification was performed on AS and healthy valves with DeCyder software v6.5 (GE Healthcare) and the multivariate statistical module EDA (Extended data analysis). The Differential in-gel analysis (DIA) module co-detected the 3 images of a gel (the internal standard and the two samples), measured the spot abundance in each image, and expressed these values as Cy3/Cy2 and Cy5/Cy2 ratios. These DIA datasets were then analysed using the Biological Variation Analysis module (BVA), which enabled the spot maps to be matched and the Cy3/Cy2 and Cy5/Cy2 ratios to be compared. Only protein spots with >1.5-fold differences in abundance were considered for the analysis. A statistical analysis was then carried out to determine the changes in protein species, with P-values below 0.05 accepted as significant when the Students t-test was applied. The gels were then re-stained with a silver staining kit (GE-Healthcare).

A 30-min pulse labeling of mitochondrial nascent chains with ³⁵S Met-Cys (EasyTag-PerkinElmer) in cultured cells was performed as described (48 (link)). All samples were treated with Benzonase according to the manufacturer’s instructions and then mixed with gel loading buffer [186 mM tris-HCl (pH 6.7), 15% glycerol, 2% SDS, bromophenol blue (0.5 mg/ml), and 6% β-mercaptoethanol]. A 12 to 20% gradient SDS-PAGE was used to separate samples, then dried for exposure with a Phosphoscreen, and scanned with a Typhoon 9400 (GE Healthcare) for quantification. Gels were rehydrated in water and Coomassie-stained to confirm loading.

Gels were scanned immediately after electrophoresis using a Typhoon 9400 fluorescence scanner (GE Healthcare Life Sciences) with parameters recommended for 2D-DIGE experiments by the manufacturer. Image analysis and relative quantification were performed with DeCyder™ 2D Software version v7.0 (GE Healthcare Life Sciences). Coordinates of significantly differing protein spots (p≤0.05 with FDR correction, ratio≥|3| when comparing both treatments) were transferred to a pick list for further processing.

Peptide SPOT arrays were synthesized using F-moc chemistry on nitrocellulose membranes at the MIT Biopolymers facility using an Intavis AutoSpot robot. Peptide spots were cut from the membrane, hydrated in 100% methanol, transferred to TBS (50 mM Tris, pH 8.1, 100 mM NaCl, 0.01% Triton X-100) with 1% bovine serum albumin (BSA), here called blocking buffer, and incubated at room temperature for ∼10 minutes. Membranes were then incubated with 10 ml of 1 µM or 100 nM c-Myc-tagged receptor (sequences in Table S6) in TBS for 1 h at room temperature. Membranes were then rinsed 3× with blocking buffer and then incubated with anti-c-myc-Cy3 antibody (Sigma Aldrich C6594) diluted 100-fold in blocking buffer for 1 hour at room temperature. Membranes were rinsed 3× with blocking buffer and scanned on a Typhoon 9400 (GE Healthcare). Images were analyzed with ImageQuant (GE Healthcare), and Cy3 intensity at 580 nm was averaged over a circular area that was equal in size for all spots for a given membrane. Typically, 5–10 known binders and their negative controls were included.

STELA was conducted as described previously, using genomic DNA (250 pg/reaction) for telomeres at 12q (12qSTELA primer), 17p (17p6 primer) and XpYp (XpYpE2 primer), and 500 pg/reaction for the ciHHV-6-associated telomere (DR1R primer)25 (link)^,34 (link)56 (link). Individual amplicons, detected by phosphor-image analysis, were sized using the Imagequant software (Typhoon 9400, GE Healthcare) with known size markers (GeneRuler 1 kb and GeneRuler High Range DNA ladder, Fermentas), and the length of the flanking sequence was subtracted. The telomere lengths were presented as scatter plots with median and interquartile ranges. Telomere lengths in the blood and PEL samples were compared using the non-parametric Kruskal-Wallis test (GraphPad Prism; GraphPad Software Inc., CA, USA).

STELA-PCR was conducted as reported previously^{59 (link)} with some minor modifications. Each STELA-PCR contained 250–1000 pg genomic DNA in 25 μl volume. The PCRs were cycled as follows: 25 cycles of 96 °C for 20 s, 63 °C (for flanking primers XpYp E2, 17p6, 12q-STELA) or 66.5 °C (for allele-specific primers XpYp-427G/415C) for 30 s, 68 °C for 10 min. Each reaction was split into three identical 0.8% LE agarose gels that were processed into Southern blots with variant-repeat detection by hybridization to ³²P-dCTP labeled DNA probes that detect (TTAGGG)_n, (TCAGGG)_n, or (TGAGGG)_n repeats. Phosphor-image analysis was conducted using a Typhoon 9400 (GE Healthcare) and ImageQuant software. N.B. the 17p6 primer annealing site is polymorphic (presence/absence) in populations. Consequently, productive amplification with 17p6 in STELA reactions behaves as a Mendelian trait (NJR unpublished).