Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

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

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2018 Zhou, Cao, Yang, Wang, Yang, Ben, Shen, Cao, Luo and Yin.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

PMC Copyright notice

Glutathione S-transferase Pi (GSTP) phosphorylation at serine-184 is crucial for its nucleus translocation and interaction with high mobility group box-1 protein (HMGB1). (A) The phosphorylation profiles of GSTP in RAW264.7 cells untreated (upper) and treated (bottom) with lypopolysaccharide (LPS; 500 ng/ml) for 30 min were detected by MALDI-QIT-TOF-MS. (B,C) GSTP WT, GSTP S184A, GSTP S184D, or empty vector was transfected into cells, and after 36 h, cells were stimulated with or without LPS (500 ng/ml) for 6 h. (B) Cytoplasmic and nuclear fractions were subjected to Western blot using Flag-GSTP and HMGB1 antibodies. (C) Cells were incubated with anti-Flag antibody and then incubated with Alexa flour488-conjugated secondary antibody (green). The nuclei were counterstained with DAPI (blue). The location of GSTP was observed under a confocal laser microscope. Scale bar: 10 µm. (D,E) Primary peritoneal macrophages (D) and RAW264.7 cells (E) were transfected with GSTP WT, GSTP S184A, GSTP S184D, or empty vector, and then were stimulated with or without LPS (500 ng/ml) for 18 h. HMGB1 level in the culture medium was determined by ELISA. (F) RAW264.7 cells were transfected with GSTP WT, GSTP S184D, GSTP S42A, GSTP S42D, GSTP Y198F, GSTP Y198D. After 36 h, cells were incubated with or without LPS (500 ng/ml) for 6 h. Nuclear and cytoplasmic fractions were analyzed by Western blot using Flag-GSTP and HMGB1 antibodies. (G) RAW264.7 cells transfected with GSTP WT, GSTP S184D, GSTP S42A, GSTP S42D, GSTP Y198F, GSTP Y198D were incubated with or without LPS (500 ng/ml) for 18 h, and then levels of HMGB1 in the culture medium were measured by ELISA. (H) HEK293 cells were transiently transfected with Flag-HMGB1, and cell extracts were incubated with wild-type His-GSTP or its mutants immobilized on Ni²⁺–IDA–agarose beads for 8 h, followed by immunoblotting with anti-His or anti-Flag antibody. (I) HEK293 cells were transfected with wild-type Flag-GSTP or its mutants and then incubated with His-tagged HMGB1. Data information: In (B,F), data are presented as mean ± SD. *P<0.05; **P<0.01 versus corresponding nuclear group by unpaired Student’s t-test. ^#P<0.05; ^##P<0.01 versus corresponding cytoplasmic group by unpaired Student’s t-test. In (D,E,G), 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.

Glutathione S-transferase Pi (GSTP) phosphorylation at serine-184 is crucial for its nucleus translocation and interaction with high mobility group box-1 protein (HMGB1). (A) The phosphorylation profiles of GSTP in RAW264.7 cells untreated (upper) and treated (bottom) with lypopolysaccharide (LPS; 500 ng/ml) for 30 min were detected by MALDI-QIT-TOF-MS. (B,C) GSTP WT, GSTP S184A, GSTP S184D, or empty vector was transfected into cells, and after 36 h, cells were stimulated with or without LPS (500 ng/ml) for 6 h. (B) Cytoplasmic and nuclear fractions were subjected to Western blot using Flag-GSTP and HMGB1 antibodies. (C) Cells were incubated with anti-Flag antibody and then incubated with Alexa flour488-conjugated secondary antibody (green). The nuclei were counterstained with DAPI (blue). The location of GSTP was observed under a confocal laser microscope. Scale bar: 10 µm. (D,E) Primary peritoneal macrophages (D) and RAW264.7 cells (E) were transfected with GSTP WT, GSTP S184A, GSTP S184D, or empty vector, and then were stimulated with or without LPS (500 ng/ml) for 18 h. HMGB1 level in the culture medium was determined by ELISA. (F) RAW264.7 cells were transfected with GSTP WT, GSTP S184D, GSTP S42A, GSTP S42D, GSTP Y198F, GSTP Y198D. After 36 h, cells were incubated with or without LPS (500 ng/ml) for 6 h. Nuclear and cytoplasmic fractions were analyzed by Western blot using Flag-GSTP and HMGB1 antibodies. (G) RAW264.7 cells transfected with GSTP WT, GSTP S184D, GSTP S42A, GSTP S42D, GSTP Y198F, GSTP Y198D were incubated with or without LPS (500 ng/ml) for 18 h, and then levels of HMGB1 in the culture medium were measured by ELISA. (H) HEK293 cells were transiently transfected with Flag-HMGB1, and cell extracts were incubated with wild-type His-GSTP or its mutants immobilized on Ni²⁺–IDA–agarose beads for 8 h, followed by immunoblotting with anti-His or anti-Flag antibody. (I) HEK293 cells were transfected with wild-type Flag-GSTP or its mutants and then incubated with His-tagged HMGB1. Data information: In (B,F), data are presented as mean ± SD. *P<0.05; **P<0.01 versus corresponding nuclear group by unpaired Student’s t-test. ^#P<0.05; ^##P<0.01 versus corresponding cytoplasmic group by unpaired Student’s t-test. In (D,E,G), 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.