Sikyungbanha-Tang Suppressing Acute Lung Injury in Mice Is Related to the Activation of Nrf2 and TNFAIP3

Figure 4

Activation of Nrf2 by SKBHT. (a) RAW 264.7 cells were treated with indicated amounts of SKBHT for 16 h or sulforaphane (5 M) as a positive control for the activated Nrf2. Nuclear proteins were extracted and analyzed by immunoblotting for the nuclear, active form of Nrf2. The membrane for Nrf2 immunoblotting was stripped and reblotted for lamin B as a loading control. Shown is representative of three independent analyses. (b) Total RNA was analyzed by qPCR for GCLC, HO-1, and NQO-1. Data represent the mean ± SEM of three independent measurements. P, P, and P were less than 0.001, compared with untreated controls (post-ANOVA comparison with Tukey’s post hoc test). (c) HEK 293 cells were transfected with plasmids encoding V5-Nrf2, HA-Ub, and Keap1 for 48 h The transfected cells were treated with or without indicated amounts of SKBHT for 16 h V5-Nrf2 was precipitated by an anti-V5 antibody and analyzed by an anti-HA antibody to reveal the ubiquitinated V5-Nrf2. V5-Nrf2 and tubulin in cell lysate were immunoblotted for inputs.

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