Nrf2 c 20

For experiments studying the Keap1-Nrf2 pathway, cells were cultured in 6-well plates and pre-treated with the indicated concentrations of tBHQ or 25 µM MG-132 for 4 hr. For MAPK and NFκB activation experiments, stable-transfected RAW264.7 cells were cultured in 6-well plates and starved in serum-free medium containing 5 mM NAC for 16 hr. Cells were subsequently induced with RANKL (200 ng/mL) and M-CSF (100 ng/mL) for the indicated times. Western blotting was performed as described previously (Yuan et al., 2016 (link)). The primary antibodies used in this study were as follows: Nrf2 (H-300) and Nrf2 (C-20) (1:100, Santa Cruz Biotechnology, Dallas, TX, USA), Keap1 (E-20) (1:100, SCBT), phospho-p38 (Thr180/Tyr182) (1:1000, Cell Signaling Technology), p38 (1:1000, CST), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:1000, CST), ERK1/2 (1:1000, CST), phospho-NFκB p65 (Ser536) (1:1000, CST), NFκB p65 (1:1000, CST), and β-actin (1:4000, SCBT). Densitomety analysis was performed using ImageJ (Schindelin et al., 2012 (link)) and normalized to the β-actin signal. Relative phosphorylation of was presented as the ratio between the phosphorylated normalized to the non-phosphorylated/total protein. NAC, tBHQ, and tBHP were obtained from Sigma-Aldrich (St. Louis, MO, USA), and MG-132 was obtained from Selleck Chemicals (Houston, TX, USA).

Cells were lysed in passive lysis buffer (Promega) and harvested with a cell scraper. Cell debris was removed by centrifuging the cell lysate at 13,000 rpm for 10 minutes at 4°C, and 30 μg of proteins were loaded on 10% SDS-PAGE gels and separated by gel electrophoresis. Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and blocked for 1 h with 10% nonfat milk in Tris-buffered saline with 0.1% Tween 20. Proteins were then blotted with antibodies against Nrf2 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA), HO-1 (H-105, Santa Cruz Biotechnology), lamin B1 (A-11, Santa Cruz Biotechnology), cleaved caspase-3 (9661, Cell Signaling, Beverly, MA), and beta-actin (C4, Santa Cruz Biotechnology). Detection of the primary antibody was accomplished using HRP-conjugated anti-mouse IgG (1 : 3000, Santa Cruz Biotechnology) and anti-rabbit IgG (1 : 3000, Santa Cruz Biotechnology). Intensities of the protein bands were evaluated by densitometric analysis using GeneGnome XRQ (Syngene Corp., Cambridge, UK).

For detection of protein expression in total cell lysates, cells were lysed in RIPA buffer (50mM Tris/Cl pH 8, 150mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS with Halt Protease inhibitor (Thermo Fisher)). Protein concentrations were measured with Bradford (Amaresco), then prepared with 5X sample buffer (0.25M Tris/Cl pH 6.8, 10% SDS, 50% glycerol, 0.5M DTT, 0.25% bromophenol blue), electrophoresed through Bolt 4–12% bis-tris polyacrylamide gels in MOPS running buffer (Thermo Fisher), transferred to nitrocellulose membranes, and subjected to immunoblot analysis. Antibodies used include: GFP GF28R (Thermo Fisher), FLAG M2 (Sigma), NRF2 H-300 (Santa Cruz), NRF2 C-20 (Santa Cruz), DDB1 A300-462 (Bethyl), CUL4A 113876 (GeneTex), KEAP1 ab66620 (Abcam), Actin A5441 (Sigma), Tubulin 21485 (CST), Ubiquitin 1859660 (Thermo Fisher).