Fig. 3

Effects of LCN2-mediated ROS on formation of the PLC in BMDCs. A BMDCs derived from WT and LCN2^−/− mice were infected with Mtb at an MOI of 1 for the indicated time periods. B BMDCs derived from WT and LCN2^−/− mice were preincubated with NAC (0.2 or 0.5 mM) for 30 min, then infected with Mtb at an MOI of 1 for 48 h. Western blotting was performed using antibodies against CRT, ERp57, ERAP1, and calnexin. C–E BMDCs derived from WT and LCN2^−/− mice were infected with Mtb at an MOI of 1, then incubated with recombinant LCN2 (1 or 5 µg/ml) for 48 h. C Western blotting was performed using antibodies against CRT, ERp57, ERAP1, and calnexin. Western blot bands corresponding to each protein were quantified, and the intensity of each target protein was normalized to the intensity of the β-actin loading control. The normalized ratio of the WT control (WT unstimulated control (UN) or Ra-only infected WT control) was set as 1.0 to compare target protein abundance in different samples. The normalized ratio is shown at the bottom of the blots. A representative blot from three independent experiments is shown. D The expression of MHC class I molecules was analyzed by flow cytometry on CD11c⁺-gated BMDCs. Data are shown as mean ± SD (n = 8). E Levels of intracellular ROS were determined by flow cytometry using ROS-ID Total ROS detection kit (1 µM, 37 °C, 30 min) in CD11c⁺‐gated BMDCs. Data are shown as mean ± SD (n = 10). Statistically significant differences were determined by Mann–Whitney test (non‐parametric unpaired two-tailed t-test; *) or Wilcoxon signed-rank test (non‐parametric paired two-tailed t-test; ^#). *p < 0.05, **p < 0.01, and ***p < 0.001; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001