pbabe retroviral vector full-length complementary dna encoding egfr gene  (Addgene inc)

Journal: Burns & Trauma

Article Title: p53 promotes the expansion of regulatory T cells via DNMT3a- and TET2- mediated Foxp3 expression in sepsis

doi: 10.1093/burnst/tkad021

Figure Lengend Snippet: Foxp3 expression is regulated by DNMT3a and TET2. ( a , b ) C57BL/6 mice were subjected to sham (n = 5) or CLP (n = 10) procedure and sacrificed after 24 h. Total mRNAs were extracted from splenetic CD4 + T cells and subjected to qPCR analysis of DNMT1, DNMT3a, DNMT3b, TET1, TET2 and TET. β-Actin was used as the loading control. The mRNA levels were normalized to the sham group ( * p < 0.01). ( c ) Total proteins were extracted from cells and used for western blotting of DNMT3a, TET2 and β-actin. The relative expressions were quantified using ImageJ and normalized to that of the loading control ( * p < 0.01, # p < 0.01). ( d ) Jurkat T cells were transfected with lentivirus containing DNMT3a mRNA or siRNA expressing plasmids or blank vector. ( e ) Jurkat T cells were transfected with lentivirus containing TET2 mRNA or siRNA expressing plasmids or blank vector. After stable expression, cells were stimulated with PBS (control) or LPS (1 μg/ml) for 24 h. Then total proteins were extracted from cells and used for western blotting analysis of Foxp3 and β-actin. The relative expression of Foxp3 was quantified using ImageJ and normalized to that of β-actin ( * p < 0.05, * * p < 0.01, # p < 0.01 vs control group in siRNA vector, ## p < 0.01 vs LPS group in siRNA vector). CLP cecal ligation and puncture, m RNA messenger ribonucleic acid, siRNA small interfering ribonucleic acid, qPCR quantitative real-time polymerase chain reaction, DNMT DNA-buffered saline, LPS lipopolysaccharide, Foxp3 forkhead/winged helix transcription factor p3

Article Snippet: Lentivirus vectors expressing the DNA fragments encoding GFP-tagged full-length DNA methyltransferase enzyme (DNMT) 3a, DNMT3a mRNA-targeted small interfering ribonucleic acid (siRNA), ten–eleven translocation 2 (TET2) and TET2 mRNA-targeted siRNA were designed, constructed, packed and purified by Obio Technology Co. Ltd (Shanghai, China).

Techniques: Expressing, Control, Western Blot, Transfection, Plasmid Preparation, Ligation, Real-time Polymerase Chain Reaction, Saline

Journal: BMC Molecular Biology

Article Title: USF2 inhibits C/EBP-mediated transcriptional regulation of the RIIβ subunit of cAMP-dependent protein kinase

doi: 10.1186/1471-2199-3-10

Figure Lengend Snippet: USF2 inhibits induction of the RIIβ promoter by C/EBP. The RIIβ promoter construct (-395 to -123) in the CAT reporter vector was co-transfected with 500 ng of expression vectors for USF1 (1), USF2a (2A) or C/EBPβ Lap (horisontally striped bars) or Lip (hached bars) isoforms or combinations of these factors. Empty expression vector was added to ensure a total of 2 μg of DNA transfected in each well. Data represent reporter activities (CAT/Luc) relative to transfection with the RIIβ promoter construct under unstimulated conditions, and were normalized for expression of luciferase from a cotransfected control vector (pGL3Control). Three separate transfections were performed in triplicate.

Article Snippet: C/EBPβ LAP (full-length) and LIP (DNA-binding domain) expression vectors created in pCMV™3 (Stratagene, La Jolla, CA) were appreciated gifts from Dr. Shizua Akira (Hyogo College of Medicine, Hyogo, Japan).

Techniques: Construct, Plasmid Preparation, Transfection, Expressing, Luciferase