β actin

Samples for western blot assays were prepared as described previously^{3 (link)}. The following primary antibodies were used: Rad51 (diluted 1:2,000; Cat. No. sc-8349; SCBT); Rad54 (diluted 1:2,000; Cat. No. sc-374598; SCBT); PCNA (diluted 1:2,000; Cat. No. sc-56; SCBT); Plk1 (diluted 1:3,000; Cat. No. sc-17783; SCBT); Oct3/4 (diluted 1:3,000; Cat. No. sc-5279; SCBT); Chk2 (diluted 1:3,000; Cat. No. sc-9064; SCBT); α-Tubulin (diluted 1:5,000; Cat. No. sc-8035; SCBT); β−Actin (diluted 1:10,000; Cat. No. ab8226; Abcam); Cyclin A (diluted 1:2,000; Cat. No. sc751; SCBT); CENP-F (diluted 1:1000; Cat. No. ab5; Abcam); γH2AX (diluted 1:2,000; Cat. No. 2577; CST); TopBP1 (diluted 1:3,000; Cat. No. sc-271043; SCBT); and Exo1 (diluted 1:1,000; Cat. No. MS-1534; Thermo Fisher). Immunoblot signals were developed with a WEST-ZOL detection system (Cat. No. 16024; iNtRON Biotechnology). The relative amount of each protein was quantified using ImageJ software.

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).

The mice were anesthetized under isoflurane (5% isoflurane, 95% O2) and perfused with artificial cerebrospinal fluid (aCSF) solution. The hippocampus was isolated and homogenized for 10 min with lysis buffer (1% Triton X-100, 0.32 M sucrose in HEPES solution) with Halt protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, CA, USA). Protein concentration was measured by BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, CA, USA). The same amounts of protein were loaded onto a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred onto 0.45-μm size pore polyvinylidene fluoride membranes. Blocking procedure was done with 5% non-fat dry milk plus 0.1% Tween 20 for 2 h, and immunoblotted with primary antibody of PKA (1:500, Thermo Fisher Scientific, Waltham, CA, USA) and β-actin for normalization (1:3000, SCBT, Dallas, TX, USA). All full blots are represented in the Supplementary Figs. S7 and S8.

SW1353 chondrocytes were collected to extract proteins, which were quantified by the bicinchoninic acid (BCA) method. After separation using a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE), proteins were moved to the polyvinylidene fluoride (PVDF) membrane, followed by loading with primary antibodies against type II collagen (1:800, Cat#28459-1-AP, Proteintech, USA), Aggrecan (1:1000, Cat#28971, Cell Signaling Technology, USA), SOX-9 (1:800, Cat #82630, Cell Signaling Technology, Boston, USA), or β-actin (1:5000, Cat#sc-69879, SCBT, USA) for 12 hours at 4° C. Then, the secondary antibody (1:2000, SCBT, USA) was introduced and cultured for 90 min, followed by exposure to ECL solution. The Bio-Rad Quantity One software was used for the quantitative analysis of bands.

Palatal fibroblasts were serum-starved and then treated with Emdogain for 30 minutes. Cell extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Binding of the antibody raised against phospho-smad3 (both Cell Signaling Technology, Danvers, MA, USA) and β-actin (SCBT) were detected with the appropriate secondary antibody directly labeled with near-infrared dyes (Invitrogen) and detected with the appropriate imaging system (LI-COR Biosciences).