Fig. 2.

Pharmacologic inhibition or genetic ablation of iNOS inhibits S-nitrosylation and nuclear translocation of CLIC4 in murine macrophages. (A–C) RAW 264.7 macrophages were pretreated with (N-(3-(Aminomethyl) benzyl) acetamidine dihydrochloride) 1400 W (100 μM) for 1 h in indicated cases before treatment with LPS and IFNγ (1 μg/mL and 10 ng/mL, respectively) for 24 h. (A) Lysates were used to perform biotin switch assays. Lysate from LPS/IFNγ-treated cells was also used for a reaction that omitted biotin. Lane 3 of CLIC4 input is representative for both LPS/IFNγ-treated pull-down assays. Five percent of lysates were used as input controls and immunoblotted for CLIC4. SNO-CLIC4:CLIC4 input ratio was calculated for treatments across three independent experiments. Statistical significance was determined using a two-tailed unpaired Student's t test. 1400W versus LPS/IFN treatment has a P < 0.05 and LPS/IFN versus LPS/IFN +1400W has a P < 0.05. (B) Media from treated plates was collected and assayed for nitrite + nitrate levels in control and treated cells. (C) Cells were immunostained for CLIC4, and nuclei were stained with DAPI and visualized with confocal microscopy. (D–F) Primary mouse macrophages from iNOS knockout and wild-type mice were treated with LPS and IFNγ (1 μg/mL and 10 ng/mL, respectively) for 18 h. (D) Lysates were used to perform biotin switch assays. Lysate from LPS/IFNγ-treated wild-type cells was also used for a reaction that omitted ascorbate (“no Asc.”). Five percent of lysates were used as input controls and immunoblotted for CLIC4. (E) Cells were used for subcellular fractionation, and nuclear and cytosolic lysates were immunoblotted for CLIC4 and β-actin. (F) Macrophage cells were immunostained for CLIC4 and visualized with confocal microscopy. Nuclei were counterstained with DAPI and are pseudocolored red for better visualization.