Cst9212s

SFII was obtained from the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, South Korea) [30 (link)]. Recombinant TNF-α and IFN-γ were purchased from R&D Systems (Minneapolis, MN, USA). Primary antibodies against p-STAT1 (sc-592, 1:5000), t-STAT1 (sc-7988, 1:2000), and CTSS (sc-6503, 1:1000) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and those against p-p65 (CST3031S, 1:6000), t-p65 (CST8242S, 1:2000), p-p38 (CST9211S, 1:2000), and t-p38 (CST9212S, 1:2000) were from Cell Signaling Technology Inc. (Danvers, MA, USA). Primary antibodies against β-actin (Thermo Fisher Scientific, Waltham, MA, USA, MA5-15739, 1:2000) and horseradish peroxidase-conjugated polyclonal secondary antibodies against mouse, rabbit, or goat IgG (GeneTex, Inc., Irvine, CA, USA, 1:10,000) were also purchased. The inhibitors SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and PD98059 (MEK1 inhibitor) were purchased from Tocris (Bristol, UK); InSolution™ JAK inhibitor I and BAY 11-7082 (NF-κB inhibitor) were purchased from Calbiochem (San Diego, CA, USA).

Shikonin (MB7082; purity > 98%; Meilunbio, Dalian, China) was dissolved in DMSO to prepare a 1000 µM stock solution that was stored at − 20 °C. The concentration of DMSO in the solution did not exceed 0.2%. The RPMI-1640 cell culture medium and RPMI-1640 complete medium (1% HEPES 1 M buffer solution, 1% sodium pyruvate, and 1% glutamine) were purchased from CYTIVA (SH30809.01). Antibodies against GADD45B (DF2375, Affinity Biosciences, Jiangsu, China), PPP3CC (19653-1-AP, Proteintech, Wuhan, China) and β-actin (WH256886, ABclonal, Wuhan, China) were purchased from Affinity Biosciences (DF2375), Proteintech (19653-1-AP) and ABclonal (WH256886), respectively, whereas antibodies against p38 MAPK (CST9212S, Cell Signaling Technology, Boston, USA), JNK (CST9252S, Cell Signaling Technology, Boston, USA) and ERK1/2 (CST4695S, Cell Signaling Technology, Boston, USA) were all purchased from Cell Signaling Technology.

Two days after behavioral testing, rats were anesthetized with sodium pentobarbital (150 mg/kg, i.p.), and the vmPFC was carefully dissected. Thirty μg of proteins per lane were electrophoretically separated on SDS-PAGE gels and transferred onto PVDF membranes which were then incubated overnight at 4°C with the appropriate antibodies. Primary antibodies used included polyclonal rabbit anti-p38 MAPK (1:1000, CST-9212S, Cell Signaling Technology, Beverly, MA, United States), anti-phospho-p38 MAPK (1:500, CST-9211S, Cell Signaling Technology, Beverly, MA, United States) and anti-β-actin (1:8000) (SC-47778, Santa Cruz Biotechnology, Santa Cruz, CA, United States). The secondary antibody was horseradish peroxidase-conjugated antibody (1:5000, SC-2030, Santa Cruz Biotechnology, Santa Cruz, CA, United States). The blots were detected using an enhanced chemiluminescence detection kit (GE Healthcare, Buckinghamshire, United Kingdom) and protein band densities were quantified using Image-J software (NIH, Scion Corporation, Frederick, MD, United States). Final data were expressed as a percent difference from that of the control group.