Pnf κb luc

Luciferase assay was performed as described previously (49 (link)). BV2 cells were seeded in 24-well plates and cotransfected with pNF-κB-Luc (Addgene, 49343) and Renilla luciferase reporter plasmids (Promega, E2231) using Lipofectamine 2000 for 24 h followed by OGD/R. The luciferase activities were determined using a Dual-Luciferase Reporter Assay kit (Promega, E1910) according to the manufacturer’s instructions.

For transient expression in mammalian cells, full open reading frames (ORF) for TRIM1 and HIF1α were amplified using a SW480 cDNA library and inserted into the pCS2-EGFP and pCS2-Flag vectors. pRK5-HA-Ub-WT and the lysine mutants plasmids were maintained in our lab [35 (link)]. HRE-luc, pNF-κB-Luc, and pRL-TK reporter plasmids were purchased from Addgene. The sequences of all plasmids were confirmed by sequencing before use.
Antibodies for GAPDH (G9545) and Flag (F7425) were purchased from Sigma-Aldrich. EGFP (sc8334) antibody was obtained from Santa Cruz Biotechnology. Antibodies for HIF1α antibodies (D1S7W, #36169S), NF-κB p65 (D14E12, #8242), phospho-NF-κB p65 (Ser536) (93H1, #3033) and IκBα (44D4, #4812) were from Cell Signaling Technology. Anti-TRIM1/MID2 (68359-1-Ig) and anti-HA Epitope Tag (901501) antibodies were from Proteintech and Biolegend. DMOG was from Selleckchem. Cell culture products were from Invitrogen. The relevant chemicals in this study were obtained from Sigma-Aldrich unless stated otherwise.

Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), streptomycin and penicillin were purchased from Life Technologies (Carlsbad, CA, USA). Enzyme-Linked immunosorbent assay (ELISA) kits for IL-1β, IL-2, IL-4, IL-10, IL-12p70, TNF-α and IFN-γ were from R&D Systems (Minneapolis, MN, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from USB Corp. (Cleveland, OH, USA) and the lactate dehydrogenase (LDH) release detection kit was from Roche Applied Science (Indianapolis, IN, USA). All kits were used according to the manufacturer’s protocols. CTX monohydrate, lipopolysaccharide (LPS), concanavalin (Con)A and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, USA). YAC-1 cells, a mouse lymphoma cell line, and RAW264.7 cells, a mouse macrophage cell line, were obtained from ATCC (Manassas, VA, USA; no. ATCC^®TIB-160^TM and ATCC^®TIB-71^TM). Antibodies for β-actin, iNOS, NF-κB-p65 and secondary antibody (HRP-linked anti-rabbit IgG) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). piNOS-Luc and pNF-κB-Luc were purchased from Addgene (Watertown, MA, USA).