Anti p38

Trizol, Dulbecco's modified Eagle's medium (DMEM), and other cell-culture reagents including fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), Lipopolysaccharide, Escherichia coli 0127:138 (LPS), and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-NF-κB, anti-JNK, anti-phosphorylated JNK, anti-ERK, anti-phosphorylated ERK, anti-p38, and anti-phosphorylated p38 mouse or rabbit antibodies were purchased from BD Biosciences (San Diego, CA, USA). The polymerase chain reaction (PCR) primers of iNOS, COX-2, IL-1β, IL-6, and β-actin were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Reverse transcription and real-time PCR kits were purchased from Bio-Rad Laboratories (Hercules, CA, USA). M-PER Mammalian Protein Extraction Reagent kit and NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Scientific) were purchased from Thermo Scientific (Waltham, MA, USA).

SCC-9 and SCC-14 cells were seeded 6 × 10⁵ and 1.2 × 10⁶ onto 6 cm dish, respectively, and treated with sulforaphane. Cell lysate were collected with 50–100 μL of protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) as previously described [48 (link)]. After centrifuged at 13000 g at 4° C for 30 min. The protein lysate were separated by 12% agarose gel and transferred onto a nitrocellulose membrane then blocking with 5% non-fat milk in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) for 1 h in room temperature and overnight with first-antibodies in 4° C and second-antibodies for 1 h in room temperature. Anti-p-ERK, anti-ERK, anti-JNK, anti-p-JNK, anti-p-Akt, anti-LC3I, anti-Beclin 1, anti-p-mTOR, anti-mTOR were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Akt, anti-pp38, anti-p38 were purchased from BD Biosciences (Bedford, MA, USA). Anti-cathepsin S was purchased from GeneTex (CA, USA). The band intensities were quantified by densitometry.

Rabbit anti-CLDN1 (51-9000) and anti-CLDN2 (51-6100) antibodies were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Rabbit anti-Akt, anti-p-Akt (Ser473), PI3K p85, p-PI3K p85 (Tyr458), and anti-ERK1/2 antibodies were from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-p-Stat3 (pY705), anti-Stat3, and anti-p38 antibodies were from BD Biosciences (San Jose, CA, USA). Rabbit anti-p-Akt (Thr308), anti-p-ERK1/2, and goat anti-β-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LY and MDC were from Tokyo Chemical Industry (Chuo-ku, Tokyo, Japan). C3G, CHL, and MβCD were from FUJIFILM Wako Pure Chemical Industries (Chuo-ku, Osaka, Japan). CAN, Cyanidin, mouse anti-ZO-1 antibody, P3G, rabbit anti-p-p38 antibody, and SB203580 were from Cayman Chemical Company (Ann Arbor, MI, USA), Tokiwa Phytochemical (Sakura-shi, Chiba, Japan), Zymed Laboratories (South San Francisco, CA, USA), Toronto Research Chemicals (North York, ON, Canada), Cusabio (Houston, TX, USA), and AdipoGen (San Diego, CA, USA), respectively. All other reagents were of the highest grade of purity available.