Mouse anti p38

For Western blot, cells were lysed in cold radioimmunoprecipitation buffer (50 mM Tris–HCl pH 7.5, 1% NP-40, 150 mM NaCl, 5 mM EDTA, 0.25% NaDOC, and 0.1% SDS) supplemented with protease and phosphatase inhibitors, centrifuged at 14,000 rpm for 20 min at 4 °C, and supernatant was collected as whole cell extract. 40 μg of proteins were resolved by SDS-polyacrylamide electrophoresis in 10% to 12% gels and transferred by electroblotting to a nitrocellulose membrane.
For protein immunoprecipitation and coimmunoprecipitation experiments, cells were lysed as already reported (46 (link)). Briefly, 500 μg whole cell extract was incubated overnight with 50 μl of protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, #sc-2003) and 2.5 μg of mouse anti-SOX2 (R&D System, #MAB2018), mouse anti-p38 (Santa Cruz Biotechnology, #81621), or normal mouse IgG (Santa Cruz Biotechnology, #sc-2025). Proteins were eluted after boiling for 10 min at 95 °C in 2× Laemmli sample buffer (Bio-Rad, catalog no.: 1610747) and detected by using SuperSignal West Femto (ThermoFisher Scientific) and imaged with ChemiDocTM Imaging Systems (Bio-Rad). A list of primary antibodies used is reported in Table S5.

The RAW 264.7 macrophage cell line was obtained from ATCC (Manassas, VA, USA). Cells were incubated in Dulbecco’s modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin; GIBCO BRL, Grand Island, NY, USA) at 37 °C in a humidified incubator under 5% CO₂. L7Gn was purchased from NPBANK (National Institute for Korean Medicine Development, Gyeongsan, Korea). LPS and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). EZ-Cytox solution was from DAEIL lab (Seoul, Korea). TOPscript cDNA synthesis kit and 2X TOPsimple™ DyeMIX-nTaq were purchased from Enzynomics (Daejeon, Korea). Rabbit anti-inhibitor of κBα (IκBα), rabbit anti-phospho (p)-IκBα (Ser-32/36), mouse anti-JNK, mouse anti-p38, rabbit anti-iNOS, goat anti-COX-2, rabbit anti-IL-1 receptor-associated kinase 1 (IRAK1), mouse anti-α-tubulin, and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-p-p38 (Thr-180/Tyr-182), mouse anti-p-ERK (Thr-202/Tyr-204), rabbit anti-ERK, rabbit anti-p-JNK (Thr-183/Tyr-185), rabbit anti-transforming growth factor-β-activated kinase 1 (TAK1), and rabbit anti-p-TAK1 (Thr-184/Tyr-187) antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).

Human dental pulp stem cells (DPSCs) were purchased from Lonza, Pharma & Biotech (Cat. # PT-5025). MEM-alpha, PBS, fetal bovine serum, L-glutamine, and Antibiotic–Antimycotic were procured from Gibco™ Fisher Scientific (Waltham, MA, USA). Poly-D-Lysine coated (BioCoat™, 12 mm) round German glass coverslips were purchased from Corning™ Fisher Scientific (Cat. # 354087; Waltham, MA, USA). RIPA buffer was from Cell Signaling Technology (Danvers, MA, USA) and BDNF ELISA kit from R&D System (Minneapolis, MN, USA). Various antibodies were procured: anti-C5L2 was from BioLegend (San Diego, CA, USA), rabbit anti-BDNF from NovusBio (Centennial, CO, USA), mouse anti-STRO-1, mouse anti-p38 and rabbit anti-pp38 from Santa Cruz (Dallas, Texas, USA). Fluorescent secondary antibodies were from Life Technologies (Grand Island, NY, USA). Lipopolysaccharide (LPS) was from Sigma-Aldrich (St. Louis, MO, USA; Cat.# L5418), and chemicals were from Fisher Chemical (Nazareth, PA, USA). siRNA targeting human C5L2, siRNA control and siRNA Reagent System were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).