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. 2018 Jun 19;62(18):1700908. doi: 10.1002/mnfr.201700908

Figure 4.

Figure 4

C151 in KEAP1 is the primary sensor for PEITC, but is not required at high inducer concentrations for the stabilization of NRF2. A) Stable KEAP1‐knockout MEF cells rescued with either WT, single mutant C151S, or triple mutant C151S/C273W/C288E of mouse N‐terminally tagged HA‐KEAP1 were generated as described.38 Cells were plated in 6‐well plates at a density of 106 cells per well, and placed in a 37 °C humidified incubator in 5% CO2 in air. On the following day, cells were treated with vehicle (0.1% DMSO) or the indicated concentrations of PEITC for 3 h. B) The animal experiments were approved by the Tohoku University Animal Care Committee and were compliant with the regulations of The Standards for Human Care and Use of Laboratory Animals of Tohoku University (Sendai, Japan) and the Guidelines for Proper Conduct of Animal Experiments of the Ministry of Education, Culture, Sports, Science and Technology of Japan. Wild‐type and KEAP1C151S/C151S knock‐in mice were generated, bred, and maintained at Tohoku University. Peritoneal macrophage production was elicited by i.p. injections of 4% thioglycolate solution using the previously described method.38 Four days later, peritoneal macrophage cells were extracted, washed, plated in 6‐well plates at a density of 106 cells per well, and placed in a 37 °C humidified incubator in 5% CO2 in air. Four hours later, when the cells had adhered to the plates, they were washed twice with phosphate buffered saline (PBS) before proceeding with treatments with vehicle (0.1% DMSO) or the indicated concentrations of PEITC for 3 h. For western blot analysis, cells were lysed, proteins were separated by electrophoresis on an 8% SDS‐polyacrylamide gel, and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 10% nonfat milk at room temperature for 1 h or overnight at 4 °C, immunoblotting was performed using the following antibodies for either 1–2 h at room temperature or overnight at 4 °C: rat monoclonal NRF2 antibody [154] at a dilution of 1:100, rat monoclonal HA antibody (Roche, 3F10, CA, USA, at a dilution of 1:1000) or rat monoclonal KEAP1 antibody [154] at a dilution of 1:100. A mouse monoclonal antibody against α‐tubulin (Sigma‐Aldrich, DM1A, 1:5000–1:10000 dilution) was used as a loading control.