Prl tk renilla luciferase control reporter vector

Transfection experiments were performed using PEI (polyethylenimine). The pGL4.32[luc2P/NF-κB-RE/Hygro] vector (Promega), the ISRE-luciferase reporter (a kind gift from Dr. Koichi Nakajima, Osaka City Univ.), the pGL3-IFNβ-luciferase reporter (a kind gift from Dr. Osamu Takeuchi, Kyoto Univ.), or pGL4-IFIT1-pro, which was constructed as described^{59 (link)}, was co-transfected into HEK293T or MEF cells with the pRL-TK Renilla Luciferase control reporter vector (Promega)^{60 (link)}. At 24 h after transfection, the cells were lysed and the luciferase activity was measured with a GloMax 20/20 luminometer (Promega), using the Dual-Luciferase Reporter Assay System (Promega).

The coding sequences of the splice factors arginine rich splicing factors 1 and 2 (SRSF1 and SRSF2) were PCR amplified from HEK293T cDNA and cloned by XbaI and BamHI digestion into pEXPR-IBA103 (IBA, Göttingen, Germany) with a C-terminal Twin-Strep-tag. The primer sequences were: SRSF1-XbaI fwd: 5′- CTATATCTAGAATGTCGGGAGGTGGTGTGATTC-3′, SRSF1-nonstop-BamHI rev: 5′- CTATAGGATCCTGTACGAGAGCGAGATCTGCTA-3′ SRSF2-XbaI fwd: 5′- CTATATCTAGAATGAGCTACGGCCGCCCCCCTC-3′, SRSF2-nonstop-BamHI rev: 5′-CTATAGGATCCAGAGGACACCGCTCCTTCCTCTTC-3′. All constructs were verified by DNA sequencing (Seqlab, Göttingen, Germany). Transient transfections were performed as described above. 200 ng of expression plasmid DNA and 200 ng of the AGA minigene construct were cotransfected. For reporter gene assays, this was done together with 50 ng of pRL-TK Renilla luciferase control reporter vector (Promega, Walldorf, Germany). All further steps were as described in Section 2.2.

Neuro2a cells (5 × 10⁴ cells/well) were cultured in 24-well plates overnight, and then co-transfected with the TDP-43 expression vector, the LUBAC expression vector, the pGL4.32 [luc2P/NF-κB-RE/Hygro] vector (Promega), and the pRL-TK Renilla Luciferase control reporter vector (Promega) using Lipofectamine 2000. The medium was replaced at 4 h-post transfection. At 24 h after transfection, the cells were lysed and the luciferase activity was measured with a GloMax 20/20 luminometer (Promega) using the Dual-Luciferase Reporter Assay System (Promega).

The infectious HIV-1 clone (pNL4-3) was obtained through the AIDS Research and Reference Reagent Program, NIAID, NIH, USA. The let-7i promoter reporter (pGL4-let-7i) was kindly bestowed by Dr. Ashish Lal (Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA). pRL-TK renilla luciferase control reporter vector was from Promega (USA). A perfect binding sequence of let-7i was inserted into the 3′ UTR of luciferase gene of the pMIR-REPORT™ miRNA Expression Reporter Vector (Promega, USA) to generate pMIR-REPORT-let-7i_bindingsite construct.
Anti-human CD3 and anti-human CD28 antibodies were from BD Biosciences (Palo Alto, CA). The anti-IL-2 and anti-CD95 antibodies for flow cytometry analysis were from BD Biosciences (Palo Alto, CA). Annexin-V cell apoptosis assay kit was from Keygen (Nanjing, China).
The let-7i mimics and negative control (mm-NC) were purchased from RiboBio (Guangzhou, China). let-7i mimic sequence: 5′-UGAGGUAGUAGUUUGUGCUGUU-3′; NC mimic sequence: 5′-UUUGUACUACACAAAAGUACUG-3′. The let-7i antisense inhibitor and control were purchased from GenePharma (Shanghai, China). Small interfering RNAs (siRNAs) smart pool used to specifically target human IL-2 mRNA were synthesized by RiboBio (Guangzhou, China).