Before treatment with recombinant human IFN-�� (1000 U/ml; sellectchem Roche, Basel, Switzerland) and/or spermidine (100 ��M; Merck, Darmstadt, Germany), 5��105 cells were seeded for 2 days in 24-well plates (Techno Plastic Products AG, Trasadingen, Switzerland). Preparation of Whole Cell Lysates After treatment, THP-1 cells were kept on ice. Cells were washed twice with phosphate buffered saline (PBS) and lysed in M-Per Mammalian protein extraction reagent? (Pierce Biotechnology, Rockford, IL) supplemented with the protease inhibitor cocktail tablets ��Complete mini?�� (Roche) for 30�C45 min. Cells were then centrifuged for 10 min at 13,000 g, and supernatants were collected. Protein concentration was quantified by UV280 nm using a NanoDrop ND1000 (Thermo Scientific, Waltham, MA).
RNA Isolation and Real-time Polymerase Chain Reaction Total RNA was isolated using an RNeasy? Mini Kit (Qiagen, D��sseldorf, Germany) and a QIA-Cube automated sample preparer (Qiagen). RNA concentration was determined by UV260 nm using a NanoDrop ND1000 (Thermo Scientific). cDNA synthesis was performed using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies Ltd). Real-time PCR was performed using TaqMan Gene Expression Assays (Life Technologies Ltd) and TaqMan Fast Universal PCR Master Mix No AmpErase UNG (Life Technologies Ltd) on a 7900 HT Fast Real-Time PCR System with SDS 2.2 Software (Life Technologies Ltd). Measurements were performed in triplicates, using the gene for human ��-actin as an endogenous control.
Phosphatase Activity Assay Phosphatase activity was assessed using the EnzChek? Phosphatase Assay Kit (Life Technologies Ltd) according to the manufacturer��s instructions and as described previously [18]. Western Blot Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Life Technologies Ltd). Membranes were blocked overnight with blocking solution (Tris-buffered saline containing 1% Tween 20 supplemented with 5% bovine serum albumin), and incubated with the diluted primary antibody (concentrations according to the manufacture?s instructions) in blocking solution for an appropriate time. Membranes were washed with washing solution (blocking solution without bovine serum albumin) for 1 h, and then incubated with horseradish peroxidase (HRP)-labelled secondary anti-mouse-, anti-goat- or anti-rabbit-IgG-antibody (Santa Cruz Biotechnology, Inc.
, Santa Cruz, CA) diluted (13000) in blocking solution for up to 30 min. Finally, membranes were washed for 1 h with washing solution and immunoreactive proteins were detected using an enhanced chemiluminescence detection kit (GE Healthcare, Little Chalfont, Cilengitide UK). Densitometric analysis of Western blots was performed using the National Institutes of Health (NIH) Image software.