Fig. 3. LPS decreases FBXL19 stability.

(A and B) C57/BL6 mice were intratracheally administered with P. aeruginosa strain PA103 (10⁴ colony-forming units per mouse). (A) Twenty-four hours later, lung tissues were analyzed by Western blotting with antibodies against FBXL19 and β-actin. i.t., intratracheal. (B) The relative amounts of FBXL19 were determined by densitometric analysis with ImageJ software. Data are means ± SEM of 6 (control) and 11 (PA103) mice. P values were calculated by unpaired Student t test. (C and D) MLE12 cells were treated with DMSO (dimethyl sulfoxide), MG132 (20 μM), or leupeptin (100 μM) for 1 hour before they were treated with LPS (10 μg/ml) for an additional 0, 6, or 9 hours. (C) Cell lysates were analyzed by Western blotting with antibodies against FBXL19 and β-actin. (D) The relative amounts of FBXL19 were determined by densitometric analysis with ImageJ software. Data are means ± SEM of three independent experiments. P values were calculated by two-way ANOVA and post hoc Tukey’s test. (E and F) HBEpCs were treated with LPS (10 μg/ml) for 6 hours. (E) Cell lysates were subjected to immunoprecipitation with an anti-FBXL19 antibody and Western blotting with an anti–K48-ubiquitin antibody. Input lysates were analyzed by Western blotting with antibodies against CBP, FBXL19, and β-actin. (F) The relative amounts of FBXL19 were determined by densitometric analysis with ImageJ software. Data are means ± SEM of three independent experiments. P values were calculated by unpaired Student t test. (G) MLE12 cells were treated with LPS (10 μg/ml) for 1, 5, or 20 hours. Total RNAs were extracted and fbxl19 mRNA abundance was measured by RT-PCR analysis. Data are means ± SEM of three independent experiments. P values were calculated by one-way ANOVA and post hoc Tukey’s test. All Western blots are representative of at least three independent experiments.