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. 2014 Apr 17;15(4):6592–6608. doi: 10.3390/ijms15046592

Figure 3.

Figure 3.

Effect of A20 on LPS-induced NF-κB signaling in RPMCs. (A) Cells were pre-transfected with pGEM-T easy-A20 or pGEM-T easy for 24 h. Near confluent cells were incubated with serum-free medium for 24 h to arrest and synchronize cell growth and then exposed to LPS (1 μg/mL) for 1 h. Protein lysates were prepared and subjected to immunoblotting with antibodies against phospho-IκBα or IκBα. Western blots are representative of three independent experiments. Bar graph shows the expression of phospho-IκBα/IκBα. Data are presented as the mean ± SD (n = 3). * p < 0.01 vs. control; # p < 0.01 vs. A20+; (B) Inhibitory effect of A20 on LPS-induced NF-κB activation in RPMCs. RPMCs were pre-transfected with pGEM-T easy-A20 or pGEM-T easy for 24 h. Near confluent cells were cultured in serum-free medium for 24 h to arrest and synchronize cell growth and then exposed to LPS (1 μg/mL) for 1 h. The DNA-binding activity of NF-κB p65 in nuclear extracts of RPMCs was measured using a NF-κB p65 transcription factor assay kit. Results were obtained from three independent experiments. Data are presented as the mean ± SD (n = 3). * p < 0.01 vs. control; # p < 0.01 vs. A20+.