P nf κb 3033

Mouse RAW264.7 macrophages and human THP-1 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Antibodies against inducible nitric oxide synthase (iNOS, #2982), cyclooxygenase-2 (COX-2 #4842), IL-1β (#12426), NLRP3 (#15101), caspase-1 (#3866), p-NF-κB (#3033), NF-κB (#8242), p-IκBα (#2859), IκBα (#4812), p-ERK (#4377), ERK1/2 (#9102), and SOD1 (#2770) were purchased from Cell Signaling (Beverly, MA, USA). Antibodies against caspase-1 (sc-398715), IL-18 (sc-7954), and GPx1/2 (sc-30147) were purchased from Santa Cruz (Santa Cruz, CA, USA).

Cells were lysed in 1× ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) and centrifuged to discard the deposition. The protein concentration was determined using BCA kits (TakaRA, Dalian, China). A total of 35 μg proteins were separated on 10% SDS-polyacrylamide gel (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h at room temperature with 5% nonfat milk dissolved in 1× TBST and incubated with specific primary antibodies at 4 °C overnight. Then the membranes were further incubated for 2 h at room temperature with the corresponding secondary antibodies. After that, labeled proteins were visualized by an ECL kit (Invitrogen, CA, USA) and captured using Gel imaging system (BD, USA). Primary antibodies against p-NF-κB (3033), NF-κB (3032), p-IκBα, (8219), IκBα (9242) were purchased from Cell Signaling Technology (MA, USA) and against β-actin (sc-1616), COX-2 (sc-376861), Sox9 (SC-166505), iNOS (SC-7271), PARP (SC-74470) and cleaved PARP (SC-56196) were purchased from SantaCruz Biotechnology (CA, USA) and diluted following the manufacturers’ protocols.

De Man-Rogosa-Sharp (MRS) broth was purchased from MBcell (Seoul, Republic of Korea). Hexadecyltrimethylammonium bromide (HTAB), and DSS (molecular weight of 40 kDa) were purchased from Alfa Aesar (Haverhill, MA, USA). Primary antibodies for TLR 4 (sc-52962), p-IκB-α (sc-8404), TNF-α (sc-33639), caspase 1 (sc-392736), IL-1β (sc-515598), COX-2 (sc-376861), AChE (sc-373901), p-Tau (sc-12952), Aβ (sc-374527), Bcl2 (sc-509), Bax (sc-7480), and β-actin (sc-69879) were purchased from Santa Cruz Biotechnology (Dallas, CA, USA). Primary antibodies for ChAT (20747-1-AP) and iNOS (18985-1-AP) were purchased from Proteintech (Chicago, IL, USA). Primary antibodies for caspase 7 (CUB-PA05689A0Rb) was purchased from Cusabio Technology (Wuhan, China). p-NF-κB (#3033), secondary antibodies for anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA). SOD determination kit was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). FITC-dextran (4 kDa), 2-thiobarbituric acid (TBA), 2-ethylbutyric acid, standard of acetic acid, propionic acid, butyric acid, and all other chemicals used were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).