Fig. 1.

CLIC4 is highly expressed in primary macrophages (Mϕ), is S-nitrosylated, and translocates to the nucleus in response to LPS/IFNγ. (A and B) RNA from mouse hematopoietic cells (A) or mouse macrophages (B), stimulated or unstimulated, as indicated, was isolated and used for CLIC4 and iNOS real-time PCR analysis. In A, RAW and macrophages were stimulated for 6 h with LPS/IFNγ. Bars represent the mean ± SEM of three replicates. Statistical significance was determined using a two-tailed unpaired Student's t test and is indicated. (C) Whole-cell lysates from stimulated or unstimulated macrophages were used for immunoblotting with CLIC4, CLIC1, and iNOS proteins. (D and E) Primary mouse macrophages 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 to detect S-nitrosylation. Lysate from LPS/IFNγ-treated cells was also used for a reaction that omitted ascorbate (“no Asc.”). Five percent of lysates were used as input controls. Assays were immunoblotted for CLIC4. Lane 2 of CLIC4 input is representative for both LPS/IFNγ-treated pull-down assays. The SNO-CLIC4:CLIC4 input ratio was calculated for all treatments across four independent experiments. Statistical significance was determined using a two-tailed unpaired Student's t test. Control versus LPS/IFN treatment has a P < 0.01 and LPS/IFN versus LPS/IFN (no ascorbate) has a P < 0.05. (E) Macrophage cells were immunostained for CLIC4 and visualized with confocal microscopy. Inset nuclei are visualized using DAPI.