Las1000 plus chemiluminescence imaging system

Whole-cell lysates were prepared using lysis buffer (1% NP-40, 50 mM Tris-HCl pH 8.0, 100 mM sodium fluoride, 30 mM sodium pyrophosphate, 2 mM sodium molybdate, 5 mM EDTA, and 2 mM sodium orthovanadate) containing protease inhibitors (10 μg ml⁻¹ aprotinin, 10 μg ml⁻¹ leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Frozen tumour samples were diced into small pieces in cold lysis buffer on dry ice and homogenised using a Tissue Tearor (Model 398, Biospec Products, Inc., Bartlesville, OK, USA) for 2 s, —three to five times, on ice, and the cell lysate was then rocked overnight at 4 °C. The lysates were cleared by centrifugation at 14 000 g for 20 min at 4 °C, and lysate protein concentrations were determined using a Bio-Rad protein assay (Bio-Rad Laboratories Hercules, CA, USA). Nuclear, membrane, and cytoplasmic fraction lysates were prepared by using a Qproteome Cell Compartment Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. Electrophoresis and western blotting were performed as described previously (Rubin et al, 2001 (link)). The hybridisation signals were detected by chemiluminescence (ECL, Amersham Pharmacia Biotechnology, Marlborough, MA, USA) and captured using a FUJI LAS1000-plus chemiluminescence imaging system.