Typhoon 9400 imager

Immobilized pH gradient (IPG) strips (pH 3–10, 24 cm) were rehydrated and the prepared samples were applied with cup loading. Isoelectric focusing was performed with a Multiphor™ II electrophoresis unit (Amersham Biosciences, Little Chalfont, Bucks, UK) for 54 kVh at 20°C in the dark [19 , 20 (link)]. The strips were equilibrated for 10 min in buffer (50 mM Tris-HCl [pH 8.8], 6 M urea, 30% [v/v] glycerol, 1% [w/v] sodium dodecyl sulfate [SDS]) containing 65 mM dithiothreitol, and then for 10 min in the same buffer containing 240 mM iodoacetamide. The equilibrated IPG strips were transferred onto 24 cm × 20 cm, 12% T, 7.5% C polyacrylamide gels made between low-fluorescence glass plates. The strips were overlain with 0.5% (w/v) low-melting-point agarose in buffer (25 mM Tris-base, 0.1% SDS, 192 mM glycine) containing 0.1% bromophenol blue. The gels were run in the Ettan DALT Twelve Electrophoresis System (Amersham Biosciences) at 2 W/gel at 20°C, until the dye fronts had run off the bottom of the gels. The two-dimensional (2-D) gels between low-fluorescence glass plates were scanned directly with a Typhoon 9400 imager (Amersham Biosciences). Normalization of the three Cy™ dyes was accomplished by adjusting the maximum pixel values to 55.000 counts by changing the photomultiplier tube voltage. The images generated were exported as tagged images (Amersham Biosciences).

Cell lysates were extracted in 0.1 M NaCl, 0.01 M TriszHCl (pH 7.6), 1 mM EDTA (pH 8.0), 1 mg/ml aprotinin, and 100 mg/ml PMSF, while protein concentration was determined by a Bio-Rad protein assay. Protein (50 μg) was boiled at 95°C in sodium dodecyl sulfate (SDS) sample buffer for 5 min, electrophoresed on 7–12% SDS-PAGE gels, and transferred to polyvinyldifluoridine membranes. These were then incubated overnight at 4°C with (i) rabbit monoclonal antibody against human RUNX2 (Cell Signaling Technology, Danvers, MA, USA; #8486; 1:250), (ii) goat polyclonal antibody against human HOXA1 (R&D Systems Inc., Minneapolis, MN, USA; AF5014; 1:500), (iii) goat polyclonal antibody against human HOXD10 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-33005; 1:500), or (vi) rabbit polyclonal antibody against human Actin (Santa Cruz Biotechnology; sc-1615-R; 1:3000). Membranes were washed three times for 15 min with phosphate buffered saline (PBS) containing 0.1% Tween20 (PBST), incubated with anti-goat or anti-rabbit secondary antibody (both Santa Cruz Biotechnology; 1:5000) at room temperature for 60 min, and then washed three times for 15 min with PBST. The signals of protein bands were visualized using ECF western blotting kit (Amersham Biosciences, Piscataway, NJ, USA) and detected by Typhoon 9400 imager (Amersham Biosciences).

Chromosomes were resolved and rDNA detected as described (El Hage & Housley, 2013 (link)), with the following alterations. Cells were collected from asynchronously growing cultures in mid-log phase without condensin depletion. To resolve S. cerevisiae chromosomes, the gel was run with a 300–900 s switch time, 120° angle, 3 V/cm at 14°C for 68 h. The gel was stained with GelRed (Biotium) in TBE for 1 h, washed twice in 2× TBE for 15 min, and imaged. The gel was transferred onto an N+ Hybond nitrocellulose membrane (GE Healthcare) through capillary transfer as described (El Hage & Housley, 2013 (link)). The rDNA probe for Southern blotting was amplified from the non-transcribed spacer region two (NTS2). The probe was labelled with [α-³³P] dATP (3,000 Ci/mmol; Hartmann) using the Prime-It II Random Primer Labelling Kit (Agilent). The probe was then added to the membrane pre-hybridised in QuickHyb Hybridization solution (Agilent), and incubated at 68°C overnight. Membranes were washed twice in 2× SSC, 0.1% SDS for 15 min at room temperature, and twice in 0.5× SSC, 0.1% SDS, rinsed in 50 mM Tris–HCl, pH 7.5 and exposed overnight using a PhosphorImager screen (Amersham Biosciences), and scanned on a Typhoon 9400 Imager.

The consensus probe for T-bet (5′-GACAGCTCACACTGGTGTGGAGCAGGG-3′) was labeled with Cyanine 5 (Sigma-Aldrich) and used in binding reactions with nuclear extracts from CD4 T cells stimulated with anti-CD3 and anti-CD28 for 16 h and then restimulated with PMA and ionomycin for 30 min. For supershift reactions, anti–T-bet (H-210; Santa Cruz Biotechnology) was added after 10 min of incubation. The samples were electrophoresed on 5% polyacrylamide gels in 0.5× TBE, and the gels were scanned using a Typhoon 9400 imager (Amersham Biosciences).