Figure 4.

Glutathione S-transferase Pi (GSTP) enters the nucleus and interacts with high mobility group box-1 protein (HMGB1) in response to lypopolysaccharide (LPS) stimulation. (A,B) RAW264.7 cells (A) and THP-1 cells (B) were transfected with Flag-GSTP. Cells were then treated with LPS (500 ng/ml) for indicated hours. Nuclear and cytoplasmic fractions were subjected to Western blot using the anti-Flag antibody. (C) RAW264.7 cells were stimulated with LPS (500 ng/ml) for indicated time, and then, were incubated with anti-GSTP antibody, followed by incubating with Alexa flour488-conjugated secondary antibody (green). The nuclei were counterstained with DAPI (blue). Nuclear and cytoplasmic GSTP were observed under a confocal laser microscope. Scale bar: 10 µm. (D) RAW264.7 cells were transfected with Flag-GSTP or empty vector, and then, were stimulated with LPS (500 ng/ml) for indicated time. Nuclear extracts were subjected to immunoprecipitation with anti-HMGB1 antibody (left) or anti-Flag antibody (right) followed by immunoblotting with anti-Flag or anti-HMGB1 antibody. The whole nuclear fractions were immunoblot analyzed with anti-Flag, anti-HMGB1 and anti-Lamin B antibodies. (E) After being treated with LPS (500 ng/ml) for 8 h, cells were incubated with rabbit anti-Flag and mouse anti-HMGB1 antibodies, and then, incubated with Alexa flour488-conjugated anti-rabbit (green) and Alexa flour555-conjugated anti-mouse (red) secondary antibodies. The subcellular localization of Flag-GSTP and HMGB1 were examined by confocal microscopy. Scale bar: 10 µm. Data information: in (A,B), data are presented as mean ± SD. **P<0.01 versus untreated nuclear group by unpaired Student’s t-test. ^#P<0.05; ^##P<0.01 versus untreated cytoplasmic group by unpaired Student’s t test.