P38 antibody

Protein concentration of cells and the prefrontal cortex were determined using a BCA protein assay kit (Pierce Biotechnology, Inc.). A quantity of 20–40 μg of total proteins was loaded onto a 10–12 % gradient polyacrylamide gel, electrophoretically transferred to a polyvinylidene difluoride membrane, and probed with the following primary antibodies: TNF-α (1:1000, Santa Cruz), IL-1β (1:1000, Santa Cruz), phospho-NF-κB p65(S536) antibody (1:500, Cell Signaling Tech. MA, USA), NF-κB (1:1000, Cell Signaling), phospho-p38 antibody (1:1000, Cell Signaling), p38 antibody (1:1000, Cell Signaling), phospho-JNK antibody (1:1000, Santa Cruz Biotechnology, CA, USA), JNK antibody (1:1000, Santa Cruz Biotechnology), phospho-extracellular signal-regulated kinase (ERK)1/2 (1:2000, Cell Signaling), ERK1/2 (1:2000; Cell Signaling), OTR (1:2000, Abcam), inducible nitric oxide synthase (iNOS) (1:500, Cell Signaling), cyclooxygenase-2 (COX-2, 1:1000, Proteintech Group, Inc., CA, USA). β-actin (1:2000; Sigma-Aldrich) was used as an internal control. Secondary antibodies were horseradish peroxidase conjugated to goat/mouse anti-rabbit IgG (1:8000, Sigma-Aldrich). The membranes were developed using an enhanced chemiluminescence detection system (Pierce, Rockford, IL).

Phospho‐Akt (Ser473 and Thr308) antibody, phospho‐GSK‐3α (Ser21)/GSK‐3β (Ser9) antibody, phospho‐p38 (Thr180/Tyr182) antibody, Akt antibody, GSK‐3α/β antibody, p38 antibody, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody were purchased from Cell Signaling (Danvers, MA, USA). α‐SMA antibody was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Alexa Fluor 488 goat anti‐mouse immunoglobulin G (IgG) and 4′,6‐diamidino‐2‐phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, USA). ML221 was purchased from Sigma‐Aldrich. LY294002 was purchased from Cell Signaling.

The α-minimal essential medium (α-MEM), fetal bovine serum (FBS), acetonitrile, water, formic acid, high-capacity cDNA reverse transcription kit, and bicinchoninic acid assay (BCA) kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). TaqMan primers for c-Fos (Mm00487425_m1), nuclear factor of activated T-cells and cytoplasmic 1 (NFATc1, Mm00479445_m1), Atp6v0d2 (Mm00656638_m1), cathepsin K (Mm00484036_m1), dendritic cell-specific transmembrane proteins (Dcstamp, Mm01168058_m1), 18S rRNA (Hs99999901_s1), and universal PCR master mix were purchased from Applied Biosystems (Foster City, CA, USA). The RNeasy kit was obtained from Qiagen (Hilden, Germany). NFATc1, c-Fos, β-actin, and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the p38 antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). PC was purchased from the National Development Institute of Korean Medicine (Gyeongsan, Republic of Korea) and stored in the herbarium (voucher number #KE-1) of the Herbal Medicine Research Division. Dried PC (0.5 kg) was extracted using 70% ethanol in distilled water (3.5 L, v/v) under reflux for 3 h, and lyophilized after filtration. PC powder was stored at −20 °C until further use.