Mouse monoclonal anti tata binding protein

The following primary antibodies were used for WBs: mouse monoclonal anti-V5 (1:5000 dilution WB, ThermoFisher Scientific, cat. no. R960-25), rabbit polyclonal anti-GFP (1:1500 dilution WB, Cell Signaling, Danvers, MA, USA; cat. no. 2555), rabbit polyclonal anti-Lamin B1 (1:1500 dilution WB, Abcam, cat. no. ab16048), mouse monoclonal anti-β-tubulin (1:1000 dilution WB, Abcam, cat. no. ab7792), mouse monoclonal anti-TATA Binding Protein (TBP) (1:2000 dilution WB, Abcam, cat. no. ab818), goat polyclonal β-catenin C-18 (1:500 dilution WB, Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-1496), and mouse monoclonal anti-UBC9 (1:1000 dilution WB, BD Biosciences, Franklin Lakes, NJ, USA; cat no. 610748). Secondary antibodies used for WB (1:5000 dilution) were: horse radish peroxidase (HRP)-conjugated rabbit anti-mouse, rabbit anti-goat, and goat anti-rabbit (Zymed, ThermoFisher Scientific). All antibodies were diluted in blocking buffer (5% skim milk [Diploma], 0.02% Tween-20 in PBS except for the GFP antibody, for which the PBS was replaced with TBS [Tris-Buffered Saline; 50 mM Tris, 150 mM NaCl, pH 7.5]).

Cells were grown in T75 flasks, serum starved for 24 hours, and then incubated with either 0.1% DMSO control, TRAM-34 (200 nM), ICA-17043 (100 nM) or Ca²⁺ free media for 1 hour. Cells were detached with 0.1% Trypsin/EDTA and washed. Protein was isolated using the RIPA buffer lysis system (Santa Cruz, Germany) and total protein concentration was determined using the DC Bio-Rad protein Assay (Bio-Rad, UK). 30 μg of protein was resolved using 10% Mini-Protean TGX precast gels (Bio-Rad) and then transferred to an immunobilon-P polyvinylidene difluoride membrane, using Trans-blot Turbo transfer packs (Bio-Rad). Membranes were blocked with 5% milk and incubated with rabbit monoclonal anti-phospho-Smad2/3 (0.231 μg/ml, Cell Signalling, USA), rabbit polyclonal anti-Smad2/3 (0.0087 μg/ml, Cell Signalling), mouse monoclonal anti-TATA binding protein (TBP) (1 μg/ml, Abcam), or mouse monoclonal anti-β-actin antibody (0.2 μg/ml, SantaCruz). Protein bands were identified by horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagent (Amersham, UK). Immunolabelled proteins were visualized using ImageQuant LAS 4000 (GE Healthcare Life Sciences, UK).

Nuclear extracts were prepared from frozen livers as previously described [19 (link)]. Briefly, liver pieces were disrupted in a Dounce hand homogenizer on ice without NP-40 and nuclei pelleted at 4000 x g for 1 minute before extract preparation. Extracts from 5 mice in each group (sham-infected, CS-infected + vehicle and CS-infected + cSN50.1) were analyzed for nuclear import of SRTFs by quantitative immunoblotting using polyclonal goat anti-cFos (Santa Cruz), monoclonal mouse anti- Y701phophorylated STAT1 (Becton-Dickinson) and polyclonal rabbit anti-NF-κBp50 and anti-NF-κB p65 (Abcam) on a Licor Odyssey Infrared Imaging System. Mouse monoclonal anti-TATA binding protein (TBP, Abcam) was used to measure TBP as a nuclear loading control for normalization.