Effects of rTMD proteins on LPS-induced inflammatory mediator production and LPS-induced signaling. (A,B) rTMD proteins from Pichia and mammalian protein expression systems were preincubated with LPS before adding to RAW 264.7 cells. After a 6-hour incubation, culture media were collected for the measurement of (A) TNF-α and (B) NO. mTMD1 represents rTMD1 from mammalian protein expression system. Values are the mean plus or minus SD (n = 3). A and B, **P < .01 and ***P < .001 compared with the LPS-treated cultures; ^###P < .001 compared with the PBS group. (C) Various amounts of LPS were used to induce the TNF-α production in RAW264.7 cells, and the effects of rTMD1 were determined. Values are the mean plus or minus SD (n = 4). *P < .05, **P < .01, and ***P < .001 compared with the LPS-treated group. Western blotting was used to assay LPS-induced phosphorylation and degradation of IκBα (D), nuclear translocation of NF-κB p50 and p65 in nuclear fractions (E), ERK1/2 and p38 phosphorylation (F), and iNOS expression (G). All results are typical of those obtained in at least 3 independent experiments.