Geneporter reagent

NIH3T3 cells were transfected with pCMV-IL10 and pCMV-TGFß, respectively, for 5 h using GenePORTER reagent (Genlantis, San Diego, CA). The supernatant was removed and replaced by the respective culture medium. Cells were cultured for 72 h before analysis by real-time PCR (see 2.5).
BM-DCs were transfected with plasmid DNA as described [25 (link)]. In brief, BM-DCs (5×10⁶ cells/ml) harvested on d8 of culture were suspended in Resuspension buffer (NanoEnTek, Pleasanton, CA), and plasmid DNA (100 μg/ml) was added. Aliquots of cell suspension (100 μl) were electroporated using the MicroPorator MP-100 (VWR Life Science, Erlangen, Germany) at optimized conditions (1,450 V, one pulse of 30 ms). Transfectants (10⁶) were transferred to wells of 24-well tissue culture plates (Greiner, Frickenhausen, Germany) containing 2 ml of BM-DC medium. Twenty-four h later, transfectants were stimulated with LPS (1 μg/ml) for 24 h, for use in subsequent T cell stimulation assays.

Expression vectors (pcDNA3.1) encoding rat C/EBPα and mouse C/EBPδ were kind gifts of David Ron (NYU Medical Ctr, New York, NY) and James Dewille (Ohio State University, Columbus, OH), respectively. An expression vector (pCMV6) encoding human C/EBPβ was purchased from Origene (Rockville, MD). An expression vector for human C/EBPβ1 (pcDNA3.1hisA) that exclusively expressed the longest form of C/EBPβ due to modification of the ATG for C/EBPβ2 was kindly provided by Linda Sealy (Vanderbilt University, Nashville, TN). Nuclear extracts from cos-7 cells overexpressing various C/EBPs (α, β and δ) were prepared exactly as described by Schreiber et al. [44] (link) following transfection of cells in 100 mm dishes using 3 µg of expression vector and GenePORTER reagent (Genlantis, San Diego, CA) according to the manufacturer’s protocol. Whole cell extracts from cells overexpressing the C/EBPs were prepared by lysis in urea buffer (7 M urea, 2% IGEPAL, 5% β-mercaptoethanol and protease inhibitor cocktail), followed by 3–5 pulses (4–6 sec each) of sonication to disrupt membranes, and a 5 min high-speed microfuge spin to clear the lysates. All procedures were carried out at 4°C [45] (link).