Illustra probequant g 50 micro column

Total RNA was isolated from cells (40 mL cultures at OD_{750 nm} ∼ 0.5) using a hot phenol method according to Tyystjärvi et al. (2001) (link), except that cell pellets were resuspended in 0.3 M sucrose and 10 mM sodium acetate (pH 4.5). Samples containing 3.75 μg total RNA were denatured with glyoxal and separated on a 1.2% agarose gel in phosphate buffer and transferred to Hybond-N membrane (Tyystjärvi et al., 2001 (link)). The pilA1 and 16S rRNA genes were amplified from genomic DNA using pilA1-f/pilA1-r and rrn-f/rrn-r primers, respectively (Supplementary Table 1). DNA fragments were labeled with α-dCTP 10 mCi/mL (Perkin Elmer) using the Prime-a-Gene Labeling System (Promega, United States) according to the manufacturer’s instructions. The probes were purified using Illustra ProbeQuant G-50 Micro Columns (GE Healthcare). Membranes were prehybridized in 6 × SSC, 1 × Denhardt’s, 0.1% SDS, 100 μL/mL herring sperm DNA at 67°C for 1 h. A denatured pilA probe was added and hybridized at 67°C overnight. Membranes were rinsed twice with 3 × SSC, 0.1% SDS and then washed twice for 10 min at 60°C in 3 × SSC, 0.1% SDS and autoradiographed. The pilA probe was removed by washing the membrane three times with 0.5% SDS at 95°C for 10 min and then reprobed with the 16S rRNA probe.

Probes for miRNA-30c and U6 (loading control) were labelled using the miRNA StarFire Kit (Integrated DNA Technologies) and purified on Illustra ProbeQuant G50 Micro-columns (GE Healthcare) according to the manufacturer's protocol. 3 µg total RNA (TRIzol isolated) was separated on a 16% acrylamide gel, transferred to Hybond N+ membranes and UV-crosslinked (0.200 J/cm²). Labeled probes were hybridized for 30′ at 39°C in hybridization buffer (7% SDS, 200 mM Na₂HPO₄, pH 7.2), subsequently washed at 42°C with 2x SSPE buffer (0.36 M NaCl, 200 mM NaH2PO4, 20 mM EDTA, 0.1% DEPC, 0.1% SDS, pH 7.4) and exposed to Phospho-image film overnight.

DNA (≥10 µg) was digested overnight with appropriate restriction enzymes and run on a 0.8% agarose gel to separate fragments. Gels were subject to depurination and neutralisation before transferring DNA to Hybond-XL membrane (RPN203S, GE Healthcare Amersham) overnight by capillary action. The membrane was then washed and dried at 80 °C for 2 h. αP32–dCTP probes were labelled using Prime-It® II Random Primer Labelling Kit (300385, Agilent) and purified with Illustra ProbeQuant™ G-50 Micro columns (28-9034-08, GE Healthcare Amersham). Following pre-hybe for 1–2 h in Rapid-hyb™ Buffer (RPN1636, GE Healthcare Amersham), the membrane was hybridised with the probe for at least 5 h, washed, exposed to Fuji RX X-ray film and the film scanned on an Epson Perfection V500 Photo flatbed scanner.

Electrophoretic mobility shift assays were performed with nuclear protein lysates obtained from cellular fractionation. Oligonucleotides probes used in the assay were synthesized (Sigma) harboring the GAS‐binding region of the human IRF1 promoter (5′‐CATTTCGGGGAAATCAGGC‐3′). After annealing, oligonucleotides were end‐labeled with [γ‐³²P]‐adenosine triphosphate (3,000 Ci/mmol; PerkinElmer) using the T4 polynucleotide kinase (New England Biolabs) and further purified by gel filtration on Illustra ProbeQuant G‐50 Micro Columns (GE Healthcare). Nuclear lysates (5 μg) were normalized in 19‐μl reaction mixtures containing 20 mM HEPES, pH 7.9; 1 mM DTT; 0.1 mM EDTA; 50 mM KCl; 1 mM MgCl₂; 5% glycerol; 200 μg/ml BSA; and 1 μg poly[dI‐dC] (Sigma). The reaction was placed on ice for 20 min before incubating with ³²P‐labeled oligonucleotide probes (10,000 cpm) for 20 min at room temperature. For the supershift samples, the lysate‐DNA mixture was incubated with 2 μg of antibodies recognizing either STAT1 (Merck, CT #06‐501), STAT2 (Merck, CT #06‐502), or IRF9 (Abcam, ab126940) for 10 min at room temperature. Samples were resolved on a 4.5% native polyacrylamide gel (37.5:1) in a 0.5 × Tris‐borate‐EDTA (TBE) buffer for 6 h at 130 V at 4°C. Gels were dried for 60 min at 80°C and visualized by PhosphorImager.

DNA fragments (see S3 Table) containing oriC_Mtb [33 (link)] and rrnAPL [19 (link)] were amplified by PCR and products were gel-purified using QIAquick column (Qiagen). 250 ng of gel-purified dsDNA or 3x FLAG ssDNA oligo (see S3 Table) were labeled with T4 polynucleotide kinase (NEB) and [γ-³²P]-ATP, and unincorporated [γ-³²P]-ATP was removed using Illustra ProbeQuant G-50 microcolumns (GE Healthcare). 20,000 cpm of labeled probe, 10 μg BSA, and the indicated amounts of DciA_Mtb or DciA_Mtb^W113A were mixed with buffer (50mM Tris-HCl pH7, 150mM NaCl, 1mM EDTA, 1mM DTT) in a total volume of 12μl and incubated for 20 minutes at room temperature. Samples underwent native electrophoresis in 4–20% nondenaturing TBE polyacrylamide gels (Invitrogen). The gels were dried and exposed to film for detection by autoradiography.