ide membranes. The membranes were then incubated with the indicated primary antibody followed by an 7621916 HRP-conjugated secondary antibody. The antibodies are: rabbit anti-C/EBPd; p38; IKKb; A20; GAPDH; phospho-p38; actin; and HRPconjugated anti-mouse and anti-rabbit secondary antibodies. The immunoreactive bands were detected using the Western LightingH Plus-ECL. Prediction of Transcription Factor Binding Sites oPOSSUM was used to predict the transcription factor binding sites in Tnfaip3. The JASPAR CORE vertebrate database was selected for transcription factor binding site matrices, and the selection parameters were set as follows: top 10% for conserved regions, 80% matrix match, and 2000/0 for upstream/downstream sequence length. Statistical Analysis All statistical analyses were performed with SPSS 13.0. Data are presented as means 6 standard deviation from at least two separate experiments. Statistical significance was determined by Student’s t test. Unless otherwise indicated, a P value less than 0.05 was considered significant. shRNA Mediated Gene Silencing against Cebpb HEK293T packaging cells were buy Vatalanib cultured in high-glucose DMEM supplemented with 10% FBS. Transfection of 10336542 HEK293T cells was conducted using Turbofect according to the manufacturer’s instructions. The specific lentiviral shRNA constructs against Cebpb were obtained from the National RNAi Core Facility in Taiwan. Lentivirus was packaged into HEK293T cells following the guidelines of National RNAi Core Facility, and the culture supernatants containing the lentivirus were collected at 48 and 72 h posttransfection. RAW264.7 cells were infected with lentiviruses in the presence of 8 mg/ml polybrene overnight and cultured in fresh medium for another 24 h. The infected cells were then selected in medium containing 0.4 mg/ml puromycin until the uninfected cells were completely killed. Results Inhibition of NF-kB and p38 Signaling Pathways in LPSinduced Bone Marrow Derived Macrophages Since NF-kB is retained in the cytoplasm through association with inhibitor kB and degradation of IkB depends mainly on IKKb, BMDMs derived from IkkbD mice were used to identify genes regulated by NF-kB. To identify the differentially expressed genes in wt and IKKb-deficient BMDMs, BMDMs generated from wt and IkkbD mice were cultured and treated with 100 ng/ml LPS for 2, 4, and 8 hours or with medium alone as a control. RNAs extracted from these BMDMs were analyzed using an Illumina MouseRef-8 v2 Expression BeadChip, which provides 25,697 probes and targets over 19,100 unique genes. To assess the depletion of Ikkb in these IkkbD BMDMs, we examined the mRNA expression levels of Ikkb in wt and IkkbD BMDMs. As shown in Fig. 1A, western blotting showed that protein amounts of IKKb were hardly detected in IkkbD BMDMs as compared to wt BMDMs, indicating the success of Ikkb depletion. Furthermore, a pilot study in microarray analysis showed that most genes were induced by LPS at 4 h. We therefore focused on the 4-hour time point of LPS treatment in subsequent experiments. To explore which genes respond to LPS via the p38 signaling pathway, BMDMs of C57BL/6 mice were preincubated with 10 mM p38 inhibitor SB202190 for 2 h, followed by 4 h of LPS treatment. SB202190 treatment did not suppress TLR4-activated proteins upstream of p38 MAPK activity, demonstrated by phosphorylation of p38 at Thr180/Tyr182. To confirm that p38 kinase activity was inhibited by SB202190, mRNA expression levels of IL-1b and IL-6, cytok