Psv β galactosidase expression plasmid

The reporter assay for NF-κB-dependent luciferase activity was conducted as previously described by Lee et al. [21 (link)]. Microglia and HEK293T cells were each co-transfected with a NF-κB-responsive luciferase reporter plasmid that contains four κB binding sites (pNF-κB-Luc, 0.5 μg, Clontech) as well as a pSV-β-galactosidase expression plasmid (0.2 μg, Promega). After 24 h, the microglia and HEK293T cells were subjected to various experimental conditions. The resulting luciferase activities were assessed by a luminometer. The findings were normalized to β-galactosidase activity. The experiments were performed in triplicate.

According to the TargetScan database, miR-124 potentially binds to MEKK3. To construct luciferase reporter vectors, the 3′-UTR of MEKK3 cDNA fragments containing the predicted potential miR-124 binding sites were subcloned into the XhoI/NotI site of psi-CHECK™-2 Vector (Promega, Madison, WI, USA). The constructs were cotransfected into HEK293 cells along with scramble (50 nM) or miR-124 mimic (50 nM) using riboFECT™ CP as described by the manufacturer. Luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega) 48 h after transfection. Renilla luciferase activity was normalised to that of firefly luciferase.
To observe the NF-κB activity, luciferase assay was performed as previously described [33 (link)]. The cells in 12-well plates were cotransfected with 0.5 μg NF-κB-responsive luciferase reporter plasmid containing four κB sites (pNF-κB-Luc; Clontech) and 0.2 μg pSV-β-galactosidase expression plasmid (Promega). After 24 h, cells were treated with different experimental conditions, and luciferase activities were analysed using a luminometer and then normalised with β-galactosidase activity.

Emodin (purity ≥99%, HPLC-grade) was purchased from Must Biotechnology Co., Ltd. (Chengdu, China). IFN-α (recombinant human IFN-α2a) and IFN-β (recombinant human IFN-β1b) were purchased from ProSpec-Tany Techno Gene Ltd. (Shanghai, China). Bortezomib was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MG132 was purchased from Sigma-Aldrich (Shanghai, China). The pSV-β-galactosidase expression plasmid was purchased from Promega (Beijing, China).