text: Western blot analysis.
  HEK 293T cells were plated at a density of 5 x 105 cells/well in six-well culture plates (BD Biosciences) 1 d prior to transfection with the appropriate plasmid DNA (2 mug total) using FuGene 6 transfection reagent (Roche). Cells were harvested 24 h posttransfection for whole-cell lysates in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Igepal (NP-40), 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1x Complete Mini Protease Inhibitor [Roche]). Protein concentration was determined by Lowry assay (Bio-Rad, Hercules, California, United States) and colorimetric reactions were read using a VersaMax microplate reader (Molecular Devices, Sunnyvale, California, United States) at an absorbance of 750 nm. Size fractionation was performed on 20 mug of protein/sample by SDS-PAGE, and the protein was transferred to nitrocellulose or PVDF membranes and subjected to immunoblotting using the indicated antibodies. Foxp3 was detected using rabbit anti-human Foxp3 polyclonal antibody (ab4728 or ab10563; Abcam, Cambridge, United Kingdom) and anti-rabbit IgG-HRP secondary antibody (Cell Signaling Technology, Beverly, Massachusetts, United States). NF-kappaB p65 and CREB-1 were detected using rabbit anti-human polyclonal (p65) or monoclonal antibody (CREB-1; 48H2) (Cell Signaling Technology). Beta-actin was detected using a mouse monoclonal antibody (AC-15; Sigma, St. Louis, Missouri, United States). For coimmunoprecipitation analysis, cell lysates were precleared with 30 mul of protein A/G plus-agarose beads (Santa Cruz Biotechnology, Santa Cruz, California, United States) and then incubated with mouse monoclonal anti-HA antibody (6E2; 1:100; Cell Signaling Technology) and 30 mul of protein A/G plus-agarose beads overnight. The immunoprecipitates were washed four times with RIPA buffer, resuspended in SDS sample buffer, and heated at 95 degreesC for 5 min. Proteins were then treated as described for Western blot analysis. 
  