Fig. 2.

Effects of LPS treatment on lung CYP2A13 expression in vivo in a CYP2A13-humanized mouse model. (A, B, and C) LPS effect on hepatic SAP mRNA level (A) and serum (B) and lung IL-6 protein level (C) in CYP2A13-humanized mice. Mice (2-month-old female) were injected ip with 1 (A and B) or 0.5 (C) mg/kg LPS, and serum and tissues were obtained at various time points as indicated for analysis. SAP mRNA levels were normalized by levels of GAPDH. IL-6 was detected by ELISA. Data represent means ± SD (n = 4–6). *p < 0.05 and ***p < 0.001, respectively, compared with 0h; one-way ANOVA followed by Dunnett's test (A and B); or with saline group (C); Student’s t-test. (D, E, and F) LPS effect on lung CYP2A13 mRNA (D) and protein (E and F) levels in CYP2A13-humanized mice. Mice (2-month-old female) were treated with 1mg/kg LPS. Lungs were obtained at various time points for analysis. Relative CYP2A13 mRNA levels (D) (normalized by levels of GAPDH) represent means ± SD (n = 4–6); *p < 0.05, compared with 0h group; one-way ANOVA followed by Dunnett's test. For immunoblot analysis, lung microsomal proteins (10 μg/lane) were prepared from tissues pooled from four mice per group and were analyzed in duplicate using a rabbit anti-CYP2A5 polyclonal antibody. In the representative blots shown (E), recombinant CYP2A13 protein was used as a positive control and lung microsome from a Cyp2a5-knockout mouse was used as a negative control, for identification of the CYP2A13 band. The position of a nonspecific band (NS) is indicated. Calnexin was detected as a loading control. For relative CYP2A13 protein levels (F), data represent means ± SD for results from three separate analyses, normalized by the amounts of calnexin detected in the same samples. **p < 0.01 and ***p < 0.001, respectively, compared with 0h; one-way ANOVA followed by Dunnett's test.