Fig. 5.

GGOH maintains endotoxin tolerance by inhibiting expression of Malt1. A, B: Peritoneal macrophages were set up and treated with LPSs, compactin (CPN), and GGOH as indicated, two times as described in Fig. 4A. Twenty-four hours after the second treatment, nuclear extracts were subjected to immunoblot analysis with antibodies against the NF-κB family of transcription factors. The immunoblot of lysine-specific demethylase 1 (LSD1) was used as a loading control (A). Cell lysates were subjected to immunoblot analysis with an antibody against K63-linked polyubiquitin chains (B). C: Peritoneal macrophages were set up and treated with 200 ng/ml LPSs and 25 μM compactin with or without 10 μM GGOH as indicated, two times as described in Fig. 4A. Twenty-four hours after the second treatment, the amount of Malt1 mRNA was quantified by RT-QPCR as described in Fig. 3A. D: The amount of Malt1 mRNA in peritoneal macrophages subjected to the indicated treatment for the indicated time was quantified as described in Fig. 3A. E, F: On day 0, peritoneal macrophages were transfected with 0.5 μM of indicated siRNA, and seeded at a density of 1.25 × 10⁶ cells per well in a 12-well plate. Twenty-four hours later on day 1, cells were treated with LPSs and compactin two times as described in Fig. 4A. On day 3, 24 h after the second treatment, cells were harvested for quantification of indicated mRNA through RT-QPCR (E), and culture medium was collected to determine the amount of IL-1β secreted into medium through ELISA (F).