Whole cell lysates and immunoblotting were as previously described [16]. The following antibodies were employed: mouse monoclonal anti-ATP5B, rabbit monoclonal anti-JNK, mouse monoclonal anti-Cdc37, rabbit polyclonal anti-p38MAPK (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti-caspase 8, mouse monoclonal anti-caspase 9, mouse monoclonal anti-phospho-p38MAPK (T180/Y182), mouse monoclonal anti-phospho-AKT (S473), rabbit polyclonal anti-phospho-AKT (T308), mouse monoclonal anti-cytochrome c, rabbit polyclonal anti-p44/42

MAPK (ERK1/2), rabbit polyclonal anti-phospho-GSK3β (S9), rabbit monoclonal anti-caspase 3, rabbit monoclonal anti-phospho-p44/42 MAPK (ERK1/2, [T202/Y204]), rabbit monoclonal anti-NF-κB/p65, rabbit monoclonal anti-phospho-NF-κB/p65 [(S536), all from Cell Signaling Technology]; mouse monoclonal anti-GSK3β,

Dasatinib datasheet anti-PARP and anti-AKT1 (BD Biosciences, CA, USA); mouse monoclonal anti-β-actin (Sigma-Aldrich); SCH 900776 research buy mouse monoclonal anti-CK2α/α’ (1AD9) and mouse monoclonal anti-CK2β (6D5) (both from KinaseDetect); rabbit polyclonal anti-phospho-JNK [(T183/Y185), Invitrogen, Carlsbad, CA, USA]. Rabbit polyclonal anti-phospho-Cdc37 (S13) was kindly provided by Dr. Miyata, Kyoto University, Japan. Secondary antibodies goat-anti-rabbit and goat-anti-mouse, coupled to alkaline phosphatase, were purchased from Jackson ImmunoResearch, Newmarket, United Kingdom. Protein-antibody complexes were visualized by a chemiluminescent detection system using CDP-Star (Applied Biosystems, Foster City, CA, USA) substrate according to the manufacturer’s instructions. The measurement of cathepsin B activity was carried out with the cathepsin B activity fluorometric assay kit (BioVision, San Francisco, CA, USA). In brief, cells were collected by scraping, washed with cold PBS and lysed with lysis buffer. 100 μg whole cell lysate was employed for the determination of enzyme activity in the presence

of amino-4-trifluoromethylcoumarin (AFC) conjugated to the cathepsin B sequence target Ac-RR (Ac-RR-AFC, 200 μM final concentration in the assay). Fluorescence emission was measured with a fluorometer (SPEX Fluorolog F2C, NJ, USA) employing FER a 400 nm excitation filter and a 505 nm emission filter. Acquired data were processed by DataMax software (Jobin YvonTM, NJ, USA). All experiments were carried out at least three times and with triple measurements, if not otherwise stated. Standard deviation values (S.D.) are indicated in the diagrams as error bars. Statistical significance of results was calculated with the Student’s t-test (two-tailed, same variance). Statistical significance is indicated in the figure legends by P values calculated between two sets of data. A preliminary chemoluminescence-based screening of a small molecule compound library in search of novel protein kinase CK2 inhibitors, led to the identification of C11, a mixture of two individual compounds; i.e.