Typhoon 9200 phosphorimager

Cellular levels of the stringent response alarmones, i.e., ppGpp and pppGpp, were measured basically as described previously [24 (link)], with modifications. The overnight bacterial culture was grown in the MOPS (4-morpholinepropanesulfonic acid) minimal medium and then was diluted in the same medium, but with a low phosphate concentration (0.4 mM) and then cultivated to A₆₀₀ of 0.2. Then, the cultures were diluted in a 1:10 ratio in the same medium, but with addition of [³²P]orthophosphoric acid (150 μCi/mL) and grown for two generations. Next, at time zero, ITCs at 1 × MIC, or serine hydroxamate (SHX), at 1 mM was added. At specific times, samples were collected and lysed with formic acid (13 M) in three cycles of the freeze-thaw procedure. Following centrifugation, nucleotides were separated by thin-layer chromatography on PEI cellulose plates (Sigma-Aldrich, Darmstadt, Germany) in 1.5 M potassium phosphate buffer (pH 3.4). The chromatograms were analyzed using the Typhoon 9200 Phosphorimager (GE Healthcare, Uppsala, Sweden). QuantityOne (BioRad, Hercules, CA, USA) software was used for densitometry analysis.

Total RNA was extracted from Drosophila S2 cells as described above. A total of 20 μg RNA was loaded per lane on a 10% acrylamide 8 M urea gel and then transferred to a Hybond N+ nylon membrane (GE Healthcare, Chalfont St Giles, Great Britain (Frankfurt)). After UV crosslinking at 254 nm the membrane was pre-hybridized in 10 ml Church buffer (1 mM EDTA pH 8.0, 0.17% phosphoric acid, 0.5 M Na₂HPO₄, 7% SDS) for one hour at 62°C. DNA oligonucleotide probes were 5′ end-labeled with γ-³²P-ATP using T4 polynucleotide kinase (NEB) and hybridized overnight in Church buffer at 62°C. The blots were washed in 2 × SSC (300 mM NaCl, 30 mM sodium citrate) and 0.2 × SSC for 30 minutes at 62°C, dried, exposed and developed on a Typhoon 9200 phosphorimager (GE healthcare). Quantification was performed using ImageJ software.

Oligonucleotides Tt72-Cy3 (24-mer) and Tt72-blunt (24-mer) were annealed at a 1:1 molar ratio (Table S1) to give a blunt-ended double-stranded substrate. The reaction mixture (5 µL) contained 0.5 pmol of the Cy3-labeled 24-bp double-stranded oligonucleotide in Tt72 buffer and was supplemented with dNTPs brought to 0.4 mM and 100 nM Tt72 pol. The samples were incubated for 20 min at 55 °C. The reaction was stopped by adding 5 µL of formamide to each tube, followed by denaturation at 95 °C for 10 min. The quenched reaction samples were separated on a 15% polyacrylamide denaturing gel containing 8 M urea in TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA-Na₂, (pH 8.0)) and scanned using a Typhoon 9200 phosphorimager (GE Healthcare).

Double-stranded oligos (sequences listed in S2 Table) were labelled with ³²P (GE Healthcare) using Polynucleotide Kinase (Fermentas) and purified with illustra ProbeQuant G-50 Micro columns (GE Healthcare). 5–10 fmol/μl were used in binding reactions with 50 ng of recombinant protein in Bclw/BSA buffer (10% Glycerol, 10 mM Hepes pH 7.9, 70 mM NaCl, 0.2 mM EDTA, 4 mM MgCl₂, 25 mM DTT, 50 μM Zn, 0.1 mg/ml BSA and 50 ng/μl polydI-dC). Binding reactions were performed for 30–40 min at 30°C and separated on a 4% polyacrylamide gel with 0.25x TBE (89 mM Tris, 89 mM boric acid, and 1 mM EDTA) as running buffer for 1h. The gel was dried, exposed and then visualized by using Typhoon 9200 PhosphorImager (GE Healthcare). For oligo competitions, a 100-fold molar excess of unlabeled DNA fragments containing either wild-type or mutated distal AP1 sequences was included at the beginning of the reaction.