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. 2018 Feb 19;9:268. doi: 10.3389/fimmu.2018.00268

Figure 3.

Figure 3

The inhibitory effect of glutathione S-transferase Pi (GSTP) on high mobility group box-1 protein (HMGB1) release is enzymatic activity independent. (A) RAW264.7 cells were transfected with Flag-GSTP(WT), Flag-GSTP(Y7F), or empty vector and then stimulated with or without lypopolysaccharide (LPS; 500 ng/ml) for 18 h. HMGB1 levels in the culture medium were measured by ELISA. (B) Flag-GSTP(WT) or empty vector was transfected into RAW264.7 cells, and then cells were pre-treated with 6-(7-nitro-2,1,3-Benzoxadiazol-4-ylthio)-hexanol for 2 h, followed by LPS (500 ng/ml) treatment for 18 h. HMGB1 levels the culture medium were detected by ELISA. (C,D) Cells were treated as same as in (A,B), nuclear and cytoplasmic fractions were analyzed by Western blot using HMGB1 and Flag antibodies. Equal loading protein was confirmed by detecting nuclear lamin B and cytoplasmic GAPDH. Data information: In (B–D), data are presented as mean ± SD. **P<0.01; n.s., not significant (P > 0.05), versus corresponding LPS-treated group by unpaired Student’s t-test.