Figure 6. Nrf2 activation upon S .... | Rockefeller University Press

Figure 6.

$Figure 6. Nrf2 activation upon S. Typhimurium infection is regulated by RIP3. (A and B) Immunoblot analysis of necrosome activation. After S. Typhimurium infection, total cell lysates of WT and Ifnar1−/− (A) or WT and RIP3−/− BMDMs (B) were subjected to immunoblot analysis with antibodies against RIP3, phospho-RIP3 (p-RIP3), MLKL, and phospho-MLKL (p-MLKL) as markers for necroptotic pathway activation. (C) Immunoblot analysis of p62, Keap1, and Nrf2. Expression levels of p62, Keap1, and Nrf2 were analyzed by immunoblot in total cell lysates of S. Typhimurium–infected WT and RIP3−/− BMDMs. (A–C) Immunoblots are representative of three independent experiments. (D) Immunofluorescence staining of nuclear Nrf2. After 6 h of S. Typhimurium infection, WT and RIP3−/− BMDMs were immunostained for Nrf2, and subcellular localization of Nrf2 was examined by confocal microscopy. Bars, 10 µm. (E) Immunoblot analysis of nuclear Nrf2. Nuclear fractions of WT and RIP3−/− BMDMs were prepared 6 h after S. Typhimurium infection and were subjected to immunoblot analysis for Nrf2 expression. Lamin B and GAPDH were used as controls. Results are representative of three independent experiments. (F and G) RIP3-dependent expression of Nrf2 target genes. Relative mRNA expression levels of Nqo1 (F) and Gclc (G) in S. Typhimurium–infected WT and RIP3−/− BMDMs were determined by real-time PCR. Values were normalized to the amounts of mRNA in uninfected BMDMs. Data are means ± SD from three independent experiments with three replicates each. ST, S. Typhimurium. UI, uninfected. ***, P < 0.001.$

Nrf2 activation upon S. Typhimurium infection is regulated by RIP3. (A and B) Immunoblot analysis of necrosome activation. After S. Typhimurium infection, total cell lysates of WT and Ifnar1^−/− (A) or WT and RIP3^−/− BMDMs (B) were subjected to immunoblot analysis with antibodies against RIP3, phospho-RIP3 (p-RIP3), MLKL, and phospho-MLKL (p-MLKL) as markers for necroptotic pathway activation. (C) Immunoblot analysis of p62, Keap1, and Nrf2. Expression levels of p62, Keap1, and Nrf2 were analyzed by immunoblot in total cell lysates of S. Typhimurium–infected WT and RIP3^−/− BMDMs. (A–C) Immunoblots are representative of three independent experiments. (D) Immunofluorescence staining of nuclear Nrf2. After 6 h of S. Typhimurium infection, WT and RIP3^−/− BMDMs were immunostained for Nrf2, and subcellular localization of Nrf2 was examined by confocal microscopy. Bars, 10 µm. (E) Immunoblot analysis of nuclear Nrf2. Nuclear fractions of WT and RIP3^−/− BMDMs were prepared 6 h after S. Typhimurium infection and were subjected to immunoblot analysis for Nrf2 expression. Lamin B and GAPDH were used as controls. Results are representative of three independent experiments. (F and G) RIP3-dependent expression of Nrf2 target genes. Relative mRNA expression levels of Nqo1 (F) and Gclc (G) in S. Typhimurium–infected WT and RIP3^−/− BMDMs were determined by real-time PCR. Values were normalized to the amounts of mRNA in uninfected BMDMs. Data are means ± SD from three independent experiments with three replicates each. ST, S. Typhimurium. UI, uninfected. ***, P < 0.001.