Anti tata binding protein tbp

Nuclear and cytoplasmic protein lysates were isolated from MDA-MB-231 bone cells after 72 hr treatment with GANT58 (0–20 μM) using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein concentrations were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific). Protein samples (20 μg/well) were separated on a 4–20% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad) by SDS-PAGE prior to being transferred to a nitrocellulose membrane (Bio-Rad) with the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were then blocked for 1 hr in 1X TBS containing 0.1% Tween-20 and 5% w/v BSA and incubated with the following primary antibodies at 4°C overnight: anti-Gli2 (1:500, Novus Biologicals), anti-TATA binding protein (TBP) (1:1000, Cell Signaling), or anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000, Cell Signaling). Following incubation with anti-rabbit IgG HRP-linked secondary antibody (Cell Signaling) at room temperature for 1 hr, protein bands were developed by Western Lightning Plus-ECL (Perkin Elmer) and imaged on a ChemiDoc MP Imaging System (Bio-Rad). The intensity of each band was determined by densitometry using ImageJ software.

Western blotting was performed as described (8 (link)). Antibodies used were mouse anti-GILZ (G-5) (Santa Cruz Biotechnology), rabbit anti-MKP-1 (Millipore, Bedford, MA), mouse anti-β-actin (Sigma-Aldrich), anti-FoxO3a, anti-TATA-binding protein (TBP), anti-phosphorylated (p) p38, anti-pERK1/2 and total ERK, anti-pAkt and total Akt (Cell Signaling Technology, Danvers, MA) as previously described (8 (link), 30 (link)). Membrane blot density was measured by scanning using the Odyssey system (Li-Cor Biotechnology, Lincoln, NE). Densitometry ratios were normalized to appropriate total protein content and results expressed as a ratio.