Bound antibodies were detected by incubating the blots in West Pico chemiluminescent substrate (Pierce, Rockford, IL). The level of immunoreactivity was measured as peak intensity using an image capture and analysis system (GeneGnome, Syngene, UK). Hybridization with anti-��-actin was used to control equal loading and protein quality. Real-Time Reverse Transcription-PCR kinase inhibitor Pazopanib cDNA synthesis was done in a 20 ��l reaction mix starting with 1 ��g of total RNA using the reverse transcription (RT) system of Promega (Madison, WI; 42��C for 30 minutes; 99��C for 5 minutes, and 4��C for 5 minutes). Real-time RT-PCR was performed using a LightCycler (Roche Diagnostics, Mannheim, Germany), and threshold cycle numbers were determined using the LightCycler software, version 3.5.

DAPK primer sequences were sense 5��-CCTTGCAAGACTTCGAAAGGATA-3�� and antisense 5��-GATTCCCGAGTGGCCAAA-3��; the two hybridization probes were 5��-CTTAATTCTTGGCTGCAGGTTCTGTG-FL-3�� and LC 5��-Red640-GTCGGAGCTGCTGGATGAAGAGTC-ph-3��. The real-time RT-PCR was performed in a final volume of 20 ��l. The final reaction mixture contained the forward and reverse primer at 10 pmol each, the LC Red640 probe at 40 pmol, the FL probe at 20 pmol, 4 mmol/L MgCl2, and 1 �� Master Amp hybridization mix. PCR was performed under the following conditions: 95��C for 30 s, followed by 45 cycles of 95��C for 0 s, 57��C for 10 s, and 72��C for 5 seconds. We used serial dilutions of the positive control cDNA of HCT116 tumor cells to create a standard curve. PCR was performed in triplicate, and the threshold cycle numbers were averaged.

Fold induction was calculated according to the formula 2(Rt-Et)/2(Rn-En), where Rt is the threshold cycle number for the ��2-microglobulin gene in the treated cells, Et is the threshold cycle number for the experimental gene in treated cells, Rn is the threshold cycle number for the ��2-microglobulin gene in non-treated cells, and En is the threshold cycle number for the experimental gene in non-treated cells. Immunoprecipitation Cells were suspended in lysis buffer (50 mmol/L Tris-HCl pH 8, 150 mmol/L NaCl, 1% NP-40) containing 1 mmol/L phenylmethylsulfonyl fluoride and 1:100 of Protease Inhibitor Cocktail Set III (Calbiochem) at 4��C for 15 minutes, and were briefly sonicated. Cell debris was removed after centrifugation. Samples were pre-cleared at 4��C for 1 hour using protein G-Sepharose beads (GE Health care, Biosciences).

Cleared lysates were then incubated with the anti-DAPK (BD Transduction, Laboratories, Lexington, NY), anti-phospho-p38 (Cell Signaling Technology Inc.), and mouse anti-Rat IgG (DIANOVA, Hamburg) overnight at 4��C, and protein G�CSepharose beads Carfilzomib were added again overnight. The beads were washed with lysis buffer three times and one time with wash buffer (50 mmol/L Tris pH 7.5). The beads were heated at 95��C for 5 minutes. Protein separation was performed by SDS-polyacrylamide gel electrophoresis, followed by Western blot analysis.