b-actin-forward 59-TTGCCGACAGGATGCAGAA-39 b-actin-reverse 59-GCCGATCCACACGGAGTACTT-39. Immunofluorescence Cells cultured in chamber slides for 24 h at 30% confluency were fixed in 4% paraformaldehyde for 10 min. Cells were incubated in a 1:50 dilution of GADD153 or Nox4 antibodies and incubated in a humid chamber at room temperature for 60 min. The cells were washed 3 times with PBS and then stained with Cy3 or Cy2 secondary Ab (1:50 dilution) (Jackson Labs) in a humid chamber at room temperature for 40 min. Cells were washed 3 times with PBS and mounted with DAPI. Images were viewed with an epi-fluorescence microscope at 406 magnification. Ten images from each preparation were taken. For GADD153 analysis, the mean fluorescence intensity (MFI) of the nuclei of each cell was calculated using ImageJ. Western blot Cells were harvested and protein was extracted using 100 mL of cell lysis buffer supplemented with 0.3% PMSF and proteinase and phosphatase inhibitors. 100 mg of protein from each sample was size fractionated on 10% SDS PAGE gels (Invitrogen) for 60 min at 180 volts. The proteins were transferred to PVDF membrane and the membrane blocked with 5% skim milk. The PVDF was probed with an antibody directed against either GADD153 (1:200), Nox4 (1:200), RAGE (1:500), phospho-p38 MAPK (1:1000), total p38 MAPK (1:1000), or b-actin (1:1000). The membrane was washed with TBS-Tween to remove any unbound proteins and incubated with the secondary antibody, goat-anti-rabbit-HRP (1:2000). The signal was developed with SuperSignal Chemiluminescent Substrate (Pierce, Rockford, IL). Reactive Oxygen Species Assay Cells were cultured in a 96-well collagen or 3DG-collagen- coated plates for 24 h. As a positive control, cells were cultured on References 1. 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