Typhoon laser scanner

1 μg of isolated mtDNA was linearized with SacI, precipitated and dissolved in 10 mM Tris HCl pH 7.5. The DNA was hydrolyzed with 0.3 M NaOH at 55°C or digested with RNase H2 (New England Biolabs) at 37°C for 2 h. Samples were run on a 0.8% agarose alkaline gel (30 mM NaOH, 1 mM EDTA) at 25 V, 4°C for 20 h and blotted onto Hybond-N+ membrane (Amersham, GE Healthcare). Single-stranded probes were end-labelled with γ-³²P ATP using T4 polynucleotide kinase (Thermo Scientific) following the manufacturer’s protocol. Double-stranded probes were generated by labelling an approximately 500 bp PCR product with α-³²P dCTP using Prime-It II Random Primer Labeling kit (Agilent Technologies). Hybridization was for 16 h at 42°C for ssDNA probes and at 65°C for dsDNA probes. The membrane was exposed to a PhosphoImager screen and scanned in a Typhoon laser scanner (GE Healthcare). The radioactive intensity was quantified using ImageJ software and plotted on a distribution plot. The median size of alkali-treated products was determined from the distribution of the radioactivity intensity and related to the size marker that was run in parallel [7 (link)].

All measurements of YFP fluorescence (excitation at 480 nm and emission at 532 nm) were performed at room temperature using Typhoon Laser scanner (GE Healthcare). MCF-7/Luc-Akt-PH and MCF-7/B2 cells were seeded in 96-well microplates at a density of 15000 cells per well. The first fluorescent measurement was performed on the next day (Day 0) and culture media were then replaced by fresh media containing only 0.1% FBS. Cells were then cultured for 48 h in absence or presence of 1 mM insulin and various inhibitors (high insulin concentrations were used in proliferation assays, in order to compensate for insulin degradation that may occur during long-term incubations (24 or 48 h) in medium containing low amount of serum). Fluorescent measurements were carried out after 24 h (Day 1) and 48 h (Day 2). Each experimental condition was performed in triplicate. Specific YFP fluorescence of MCF-7/B2 cells was obtained by substracting the fluorescence obtained with the MCF-7/Luc-Akt-PH cells cultured in parallel under the same experimental conditions (background fluorescence). The effects of treatments on cell proliferation were obtained by dividing the specific YFP fluorescence measured on the day of the assay by the initial fluorescence (specific YFP fluorescence measured at Day 0).

The osk 3’ UTR AB probe was transcribed using MAXIscript Kit (Ambion) and uniformly labeled with [α-³²P]UTP (800Ci/mmol, Perkin Elmer). UV cross-linking assay was performed as described [11 (link)], except that 10X binding buffer consisted of 60mM Hepes pH7.9, 300mM KCl and 20mM MgCl₂, and was supplemented with protease inhibitors (Roche). ~500ng of purified recombinant Bru proteins was used. After electrophoresis of cross-linked adducts, gels were dried (Bio-Rad) and exposed to a Phosphor Screen (Molecular Dynamics) for 12 hr. The screen was then analyzed with a Typhoon laser scanner (GE Healthcare).

Plasma and serum samples from 10 healthy donors were collected by vein puncture after informed consent separately into four different S-Monevette tubes (Sarstedt) prepared with Na₃-citrate, K₃-EDTA, recombinant hirudin, or clot activator (silica). Samples of the same type were combined, aliquoted and stored at −80 °C until use. Each aliquot was thawed only once. 50 μL of plasma or serum was supplemented with TFMI-3 at zero, 100 nM, or 1000 nM final concentration added in less than 1 μl causing minimal dilution. After 10 min pre-incubation with TFMI-3 at room temperature 5 μl of Cy3-pro-FD27 (link) was added resulting in 91 μg/ml (3.5 μM) final concentration. The mixtures were incubated at 37 °C for 24 hours. Aliquots were withdrawn in every hour in the first 8–10 hours then a final sample was collected after 24 hours. Samples were immediately mixed with reducing SDS-PAGE sample buffer, heated (95 °C, 1 min) and frozen until analysis. Samples were run on 12.5% gels, and the gels were scanned with a Typhoon laser scanner (GE Healthcare). Band intensities were quantified by densitometry.