Fig. 2

TNF-α induces de novo VCAM-1 protein synthesis via TNFR1-mediated manner. a, b Cells were pretreated with various concentrations of actinomycin D (Act. D, transcription inhibitor) or cycloheximide (CHI, translation inhibitor) and then incubated with 15 ng/ml TNF-α for 16 h. The cell lysates were analyzed by Western blot using a VCAM-1 or GAPDH (as a control) antibody. c Involvement of TNFR in TNF-α-induced VCAM-1 expression, cells were pretreated with various concentrations of TNFR neutralizing antibody (TNFR nAb; 1, 3, or 10 μg/ml) for 1 h and then incubated with TNF-α for 16 h. d Cells were pretreated with Act. D (10 μM) or TNFR nAb (3 μg/ml) for 1 h and then incubated with TNF-α for 4 h. The VCAM-1 mRNA (open bar) and promoter activity (gray bar) were analyzed by real time-qPCR and promoter assay, respectively. e Cells were transfected with siRNA of TNFR1 or scramble (scrb, as a control) for 24 h and then incubated with TNF-α for 16 h. The VCAM-1 protein (a, b, c, e) was analyzed by Western blot using respective antibodies. Data are expressed as mean ± SEM of three individual experiments (n = 3). ^* P < 0.05, ^# P < 0.01 vs. TNF-α alone