The up-regulation of NF-B target genes [36,37] in our microarray are shown partly in Table 1. 7, the up-regulation of 341 genes and 250 genes were observed, while 392 genes and 119 genes were down-regulated in Thy-1 N. Expression of interleukin (IL)-1, IL-6, proliferating cell nuclear antigen, -easy muscle mass actin, collagen type IV and excretion of urinary protein was increased in rats with Thy-1 N and decreased in pyrrolidine dithiocarbamate-treated rats with Thy-1 N. These data indicated that this significant changes in the gene profile were coupled with the pathological changes of Thy-1 N, and activation of NF-B may contribute to the pathogenesis of GMCs apoptosis, proliferation, extracelluar matrix accumulation and proteinuria in Thy-1 N. transcription and microarray analysis were performed as reported previously [13]. Briefly, total RNA from rat renal cortices with or without Thy-1 N was extracted and sent to Shanghai Gene Organization for analysis. The chips were scanned by an Agilent scanner and read with Imagene software to analyse the intensities of the fluorescent signals. The data were normalized by Genespring, and the Cy3/Cy5 ratios of the two groups (Thy-1 N and control) were obtained to screen out differently expressed genes: the down-regulated genes with a ratio of lower than 05 and the up-regulated genes with a ratio Dienestrol higher than 2. Reverse transcriptionCpolymerase chain reaction The mRNA levels of renal tissue in rats with Thy-1 N at 40 min, 24 h and on day 7 after administration of Thy-1 antibody were assayed by RTCPCR. As explained above, equal amounts of total RNA (2 g) from each sample were converted to cDNA. The RT reaction was subject to PCR amplification in a 20-l reaction volume with 05 mol/l of each primer. The primer sequences were as follows: interleukin (IL)-1, forward primer, 5-CTGCAGCTGGAGAGTGTGG-3 and reverse primer, 5-CAT CCC ATA CAC ACG GAC AAC TAG-3; IL-6, forward primer, 5-AGA GGA TAC CAC CCA CAA C-3 and reverse primer, 5-GTT TCG GTC TCA GTA AGT C-3; and -actin, forward primer 5-TGA CGT TGA CAT CCG TAA AG-3 and reverse primer LHR2A antibody 5-ACA GTG AGG CCA GGA TAG AG-3. PCR was performed at 94C for 10 min followed by 28 cycles of denaturation, annealing and extension at 94C for 30 s, 58C for 30 s and 72C for 1 min, respectively, and the final extension at 72C for 10 min. PCR reactions for each sample were performed in duplicate. Amplication products were run on 1% agarose gel. Ratios for IL-1 and IL-6/-actin mRNA were calculated for each sample. Immunohistochemical examination The paraffin-embedded samples of the tissues from renal cortices (4 m) were examined to detect NF-B p65, Dienestrol proliferatiny cell nuclear antigen (PCNA), -easy muscle mass actin (-SMA) and collagen type IV (CL-IV) protein at 40 Dienestrol min, 24 h and on day 7 by indirect immunohistochemistry [14]. In order to investigate NF-B activation, we used a monoclonal antibody (anti-NF-B, p65 subunit: MAB3026; Chemicon International, Inc., Temecula, CA, USA) that recognizes specifically an epitope around the p65 subunit that is masked by bound IB. Several studies have shown that this antibody detects Dienestrol activated NF-B exclusively, because it recognizes p65 only in the absence of IB [15C17]. In brief, the sections were incubated with monoclonal antibodies of NF-B p65 (1:150), PCNA (1:75; Dako, Copenhagen, Denmark), -SMA (1:50; Dienestrol Sigma) or CL-IV (1:40; Neumarker, Fremont, CA, USA) and followed incubation with horseradish peroxidase-conjugated secondary antibody (1:500; Jackson, West Grove, PA, USA) or biotinylated anti-mouse IgG for 30 min and visualized by avidinCbiotin peroxidase reaction (Vector, Philadelphia, PA, USA). Unfavorable controls were incubated without main antibody. NF-B p65-positive and PCNA-positive cells were counted in 20 glomeruli from each section. The semiquantitative analysis on optical density (OD) value of -SMA and CL-IV staining by immunohistochemistry was completed by NYD-1000 colour picture analysis through detecting the OD value of 20 glomeruli per section densitometry under microscope. These proteins were determined by calculating the mean OD value from all specimens in the three groups. Renal histological examination.