Fig. 5.

LPS elevates GSS expression in RAW264.7 cells through the Nrf2 and NF-κB pathways. (A) RAW264.7 cells were pretreated with or without CHX (1 μg/mL) for 30 min, followed by LPS stimulation (1 μg/mL) for the indicated time. GSS level in cell lysates were detected by western blot (*P < 0.05 compared with control, #P < 0.05, ##P < 0.01, and ###P < 0.001 compared with CHX + control). (B) RAW 264.7 cells were treated with LPS (1 μg/mL) for the indicated time, and total RNA was isolated and subjected to GSS mRNA level detection by quantitative PCR. (C) RAW 264.7 cells were treated with LPS (1 μg/mL) for the indicated time, and nuclear and cytoplasmic fractions were extracted for immunoblot analysis using antibodies against Nrf2, GSS, GCLC, and GCLM. (D) RAW 264.7 cells were pretreated with Nrf2 inducer tBHQ (10 μM) (*P < 0.05, **P < 0.01, and ***P < 0.001 compared with control) or (E) were pre-incubated with Nrf2 inhibitor ML385 (5 μM) for 30 min, and than were stimulated with LPS (1 μg/mL) for 12 h. Nuclear and cytoplasmic fractions were extracted for immunoblot analysis using antibodies against Nrf2, GSS, GCLC, and GCLM (##P < 0.01 and ###P < 0.001 compared with control; *P < 0.05 and **P < 0.01 compared with LPS stimulation). (F) RAW 264.7 cells were treated with LPS (1 μg/mL) for the indicated time and analyzed by western blot using the indicated antibodies (*P < 0.05, **P < 0.01, and ***P < 0.001 compared with control). (G) RAW264.7 cells were pre-incubated with an inhibitor of IκB kinase Evo (10 μM) for 30 min and stimulated with LPS (1 μg/mL) for 15 min. Cell lysates were detected by western blot through using indicated antibodies. (I) RAW264.7 cells were pre-incubated with Evo (10 μM) for 30 min and stimulated with LPS (1 μg/mL) for 12 h. Cell lysates were detected by western blot using antibodies against Nrf2, GSS, GCLC, and GCLM (*P < 0.05 compared with LPS stimulation; ##P < 0.01 compared with control). Data represent the mean ± SD of three independent experiments.