Figure 2

Signalling pathway engaged in IL-1-dependent activation of mimitin gene. A. Representative Northern blots: HepG2 cells stably transfected with retroviral vector pCFG5-IEG2 encoding nondegradable mutant of IκBα, and cells with an empty vector (mock-control) were stimulated with IL-1 (12 h) and the level of mimitin transcript was measured. Results obtained for both types of cells were normalized to 18S rRNA level. B. The graph represents data calculated from three independent Northern blot experiments. Error bars indicate the values of standard deviation (**p < 0.02). C. HepG2 cells were pretreated with: 10 μM U0126, 10 μM SP600125, or 10 μM ZM336372 for 30 min and then stimulated with 15 ng/ml IL-1 for 12 h. The level of mRNA coding for mimitin was estimated in comparison to control (unstimulated cells) by real-time PCR. Error bars indicate standard deviation from three independent experiments (*p < 0.05, **p < 0.02). D. Western blot analysis confirming activity of inhibitors of MAPK kinases. HepG2 cells were pretreated for 30 min with inhibitors (as in Fig. 2C), stimulated with 15 ng/ml IL-1 for 10 min and then cell lysates were analyzed by Western blotting with anti-P-Erk, anti-P-JNK and anti-P-p38 antibodies. The blot was later stripped and reprobed with an α-tubulin antibody (bottom panel) to ensure equal loading.