Fla 9000 phosphorimager

In vitro transcription/translation was performed using the commercially available PURExpress system (New England Biolabs). Reactions were mixed according to the manufacturer’s recommendations by the addition of 2.2 μl of linear DNA of each construct giving a final volume of 10 μl. Polypeptide synthesis was carried out in the presence of [³⁵S]-methionine at 37°C for 15 min under 700 rpm shaking. Translation was stopped by the addition of TCA to a final concentration of 5% and incubated on ice for at least 30 min. Total protein was sedimented by centrifugation at 20,000 g for 10 min at 4°C in a tabletop centrifuge (Eppendorf, Germany). The pellet was resuspended in 2× SDS/PAGE sample buffer, supplemented with RNaseA (400 μg/ml) to digest the stalled peptidyl-tRNA, and incubated at 37°C for 15 min under 1000 rpm agitation. The samples were resolved on 12% Bis-Tris gels (Thermo Fisher Scientific) in MOPS buffer. Gels were dried on Hoefer GD 2000 dryer (Hoefer, US), exposed to a phosphorimager screen for 24 hr, and scanned using the Fujifilm FLA-9000 phosphorimager for visualization of radioactively labeled protein species.

The generated constructs were translated for 20 min. in the PURExpress Δ-Ribosome kit supplemented with high-salt-washed ribosomes isolated from HDB140, HDB143 (uL23 Δloop), or HDB144 (uL24 Δloop). Plasmid DNA of each construct (300 ng) was used as a template for polypeptide synthesis, and translation was carried out in the presence of (³⁵S) Methionine at 37°C for 20 min and shaking at 500 r.p.m. For ADR1a constructs, the translation reactions also included either 50 μM zinc acetate or 50 μM of the Zn²⁺ chelator TPEN. Translation was stopped by treating the sample with a final concentration of 5% trichloroacetic acid (TCA) and incubated on ice for 30 min. The TCA precipitated samples were subsequently centrifuged at 20,000 g for 10 min in a tabletop centrifuge (Eppendorf) and the pellet obtained was solubilized in sample buffer, supplemented with RNaseA (400 μg/ml), and incubated at 37°C for 15 min. The samples were resolved on 12% Bis-Tris gels (Thermo Scientific) in MOPS buffer for ADR1 and MES buffer for Spectrin and Titin. Gels were dried and subjected to autoradiography and scanned using the Fujifilm FLA-9000 phosphorimager for visualization of radioactively labeled translated proteins.

For each Southern blot, 3–5 μg of genomic DNA digested with Eco RV and Ssp I were loaded on a 1% agarose gel and electrophoresis was performed overnight at 1V/cm. The gel was manually transferred overnight in 20X SSC, on a Hybond-XL nylon membrane (GE Healthcare), according to manufacturer recommendations. Hybridization was performed with a 302 bp ³²P-randomly labeled CAN1 probe amplified from primers CAN133 and CAN135 (Supplemental Table S2) (22 (link)). Each probe was purified on a G50 column (ProbeQuant G50 microcolumn, GE Healthcare) and specific activities were verified to be above 2.4 × 10⁸ cpm/μg. The membrane was exposed 3 days on a phosphor screen and quantifications were performed on a FujiFilm FLA-9000 phosphorimager, using the Multi Gauge (v. 3.0) software. Percentages of DSB and recombinant molecules were calculated as the amount of each corresponding band divided by the total amount of signal in the lane, after background subtraction.

Total RNA was extracted at different time points after rifampin addition (80 µg/ml) from 5-ml samples of H. pylori cultures at an OD₆₀₀ of 0.5 to 0.9 by using a NucleoSpin miRNA kit from Macherey-Nagel (4 (link)). For Northern blotting, 5 µg of RNA was separated on an agarose-formaldehyde gel and transferred to a Hybond N+ membrane (Amersham Biosciences, Inc.) overnight by passive transfer in 10× SSC (pH 7) buffer (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Transferred RNA was fixed to the membrane via UV irradiation for 2 min. The membrane was blocked for 45 min at 65°C with ULTRAhyb hybridization buffer (Ambion), then 5 µl of ³²P-labeled riboprobe was added and the membrane was further incubated overnight at the same temperature. After three washes for 10 min at 65°C with 2× SSC, 0.2% SDS, the membrane was exposed to a phosphorimager screen (Kodak) and scanned with an FLA-9000 phosphorimager (Fujifilm).

Cleavage assays were conducted using 10 μM SNAP-25 or 1 μM syntaxin fusion protein, respectively, each 1 μL of transcription/translation mixture of the respective substrate as [³⁵S]-methionine-labeled protein, and purified LC/C derivative at 1 to 3 μM final concentrations in 10 μL. Incubation was done for 60 min at 37°C in toxin assay buffer. Reactions were stopped by the addition of an equal volume of double-concentrated sample buffer (120 mM Tris-HCl, pH 6.75, 10% (v/v) β-mercaptoethanol, 4% (w/v) SDS, 20% (w/v) glycerol, 0.014% (w/v) bromphenol blue) and then subjected to SDS-PAGE using 10% or 15% tris/glycine gels (the latter using acrylamide/bis-acrylamide in 73.5:1 ratio). Subsequently, gels were dried and radiolabeled proteins were visualized employing a FLA-9000 phosphorimager (Fuji Photo Film, Co., Ltd., Tokyo, Japan). Quantification of cleavage was done by means of the radiolabeled substrates by phosphorimaging using the Multigauge 3.2 software (Fuji Photo Film).

Time-course experiments were performed in 10 μl, containing 5 nM labelled RNA substrate, and 100 nM Dicer^SOM and Dicer^ΔHEL, respectively, in 30 mM Tris (pH 7.0), 30 mM NaCl, 1 mM DTT, and 2 mM MgCl₂ at 37°C. Increasing concentrations (12.5, 25, and 50) of Dicer^SOM and Dicer^ΔHEL1, respectively, were mixed with 5 nM labelled RNA substrate in 30 mM Tris (pH 7.0), 30 mM NaCl, 1 mM DTT, and 2 mM MgCl₂. After 60 min incubation at 37°C, the reactions were stopped with equal volume of 95% formamide, boiled for 5 min, and analyzed on a 20% polyacrylamide gel containing 8 M urea.
After electrophoresis, the gels were exposed for 6-18 hours onto a phosphor imaging screen (Fujifilm). The signal was detected using FLA 9000 phosphorimager (Fujifilm) and analyzed in Multi Gauge v3.2 software.