hat medium was supplemented with 20% conditioned medium from L929-cells containing macrophage colony stimulating factor. DNA Constructs and MS 275 site Transfection pEYFP-N1-DATG-Lifeact was constructed as follows: Lifeact cDNA, containing human codon sequences flanked by a 59 BglII and 39 EcoRI restriction site, was synthesized by GenScript Corporation and provided in a pUC57 plasmid. The Lifeactfragment did not contain a Kozak sequence, therefore, a forward primer was designed to induce a BglII site and a Kozak sequence in front of the Lifeact start codon and used together with the M13 universal reverse primer to amplify Lifeact from pUC57 by PCR. PCR products were digested with BglII and EcoRI and ligated into pEYFP-N1-DATG plasmid DNA. For transfection, cells were seeded in 6 well plates at 300 000 cells/well and incubated overnight. DNA was diluted in 1 ml serum-free DMEM and incubated for 20 minutes at 37uC with 24 ml Targefect-RAW transfection reagent. Transfection complexes were added to wells containing 2 ml fresh culture medium and incubated for 4 hours at 37uC after which medium was refreshed. A stable cell population was established by culturing cells for two weeks in medium containing 500 mg/ml G418 and cloning by limited dilution. NAMPT and NAPRT Expression NAMPT and NAPRT expression was analyzed by western blot analysis. Whole cell lysates were prepared using 5x SDS-sample buffer SDS, 25% b-mercapto-ethanol, 50% glycerol, 0.05% w/v bromophenolblue, and 312.5 mM Tris-Cl, pH 6.8) and stored at 220uC until analysis. Before lysates were loaded onto 12% SDS-PAGE gels, they were heated at 95uC for 5 minutes. After electrophoretic separation, proteins were blotted onto PVDF membranes. Membranes were blocked with 5% skimmed milk in PBS-T or TBS-T for 1 hour and labeled overnight with a-Tubulin and either a-NAMPT or a-NAPRT antibodies at 4uC. After washing the membranes three times for 5 minutes with PBS-T or TBS-T, they were incubated with IRDye secondary antibodies for one hour at room temperature. This was followed by 4 wash steps of 5 minutes each in PBS-T or TBS-T, a final wash step in PBS, and signal detection on the Odyssey Infrared Imaging System. Cell lysate from mouse PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19657107 embryonic fibroblasts was used as positive control for detection of NAPRT. Materials and Methods Reagents FK866 was obtained from Enzo Life Sciences. All other reagents were obtained from Sigma-Aldrich, unless stated otherwise. 2 NAD+ Controls Macrophage Morphodynamics NAD/NADP Measurements Intracellular NAD/NADP was measured according to the protocol published by Wosikowski et al.. Cells were seeded in 25 cm2 tissue culture flasks and incubated in either control medium or medium with 10 nM FK866 for 3, 6, 15, or 24 hours prior to sample collection. Cells were detached mechanically by scraping in fresh culture medium and counted. Suspensions of 2.06106 cells were spun down and cell pellets were resuspended in 2 ml 0.9% NaCl, split in two and kept on ice. Both fractions were spun down and NaCl was removed. One fraction was used to measure NADH and NADPH levels, and the other fraction was used to measure NAD+ and NADP+ levels. For NADH measurement, cell pellets were resuspended in 200 ml 0.02 M NaOH containing 0.5 mM L-cysteine and incubated at 60uC for 10 minutes. The alkaline lysates were then neutralized with 60 ml 0.5 M Gly-Gly buffer, pH 7.6, and kept on ice. For NAD+ measurement, cells were lysed in 200 ml ice cold 0.5 M perchloric acid and incubated at 4uC for 15