Journal: Viruses

Article Title: The Insertion of an Evolutionary Lost Four-Amino-Acid Cytoplasmic Tail Peptide into a Syncytin-1 Vaccine Increases T- and B-Cell Responses in Mice

doi: 10.3390/v15081686

Figure Lengend Snippet: Characterisation of non-fusogenic mutants of HERV-W Env. ( A ) Schematic overview of plasmids encoding an assembled HERV-W Gag (W-Gag) sequence and three different versions of the HERV-W envelope (W-Env), Syncytin-1, sequences: wildtype Env (green, HERV-W WT ), Env with LQMV insertion (purple, HERV-W LQMV ), and Env with a C>A amino acid substitution (blue, HERV-W C>A ). The Gag and Env antigens are separated by a self-cleavable P2A peptide sequence, and the expression of the antigens is controlled by a CMV promoter (CMV p.). The illustration also depicts the location of the R-peptide, the CX 6 CC and CXXC disulfide motifs, and the different domains of the Env: the surface subunit (SU) and the transmembrane subunit (TU), where the TU contains the ectodomain (Ecto), the transmembrane domain (TM), and the cytoplasmic tail (CT). The scissor illustrates the cleavage site between SU and TU. ( B ) Transmission electron microscopy (TEM) pictures of human T24 cells transfected with plasmids encoding GFP, HERV-W WT , HERV-W LQMV , or HERV-W C>A . The top row shows an overview of several cells, and the bottom row shows individual cells at a higher magnification. ( C ) ( right ) Graph showing the geometric mean fluorescent intensity (MFI) of the HERV-W Env surface expression of two technical repeats of transfected HEK293 cells, as indicated by bullets, and ( left ) histograms showing the distribution of one representative sample of each of the HERV-W plasmids and a negative control without transfection (Neg. ctrl). ( D ) The same as ( C ) but depicting results for T24 cells. ( E ) The MFI of the HERV-W Env surface expression of A549 cells 24 h after transduction with the hAd19a/64 vaccines encoding either HERV-W WT , HERV-W LQMV , or the empty expression cassette (Neg. ctrl vaccine). Bullets illustrate the three technical replicates per condition. ( F ) The Western blot shows HERV-W Gag expression in A549 cells 24 h after transduction with either the HERV-W WT or HERV-W LQMV vaccine. The right graph shows the difference in the expression of the HERV-W Gag in the Western blot between the two vaccines, relative to the expression of the housekeeping protein GAPDH.

Article Snippet: The membrane was incubated overnight at 4 °C with the primary antibodies: the anti-T2A-antibody (Crb200569d; CRB discoveries, Cleveland, UK; 1:2000) to detect the P2A peptide on HERV-W Gag and the housekeeping control protein anti-GAPDH antibody (ab181602; Abcam, Cambridge, UK; 1:8000).

Techniques: Sequencing, Expressing, Transmission Assay, Electron Microscopy, Transfection, Negative Control, Transduction, Vaccines, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Pro-Resolving Lipid Mediator Resolvin E1 Mitigates the Progress of Diethylnitrosamine-Induced Liver Fibrosis in Sprague-Dawley Rats by Attenuating Fibrogenesis and Restricting Proliferation

doi: 10.3390/ijms21228827

Figure Lengend Snippet: Effect of RvE1 on tissue levels of NF-κB and Nrf2, and molecules targets. Western blot analysis of ( A ) cytoplasmic and ( B ) nuclear NF-κB; and ( C ) cytoplasmic and ( D ) nuclear Nrf2. The cytoplasmic levels were normalized to GAPDH as housekeeping and nuclear levels were normalized to histone H1 as housekeeping. ( E , F ) serum levels of TNF-α and IL-10. ( G – I ) tissue levels of GSH, GSSG and GSH:GSSG ratio. n = 6–9 rats per experimental group. Asterisk indicates p < 0.05, and the letters identify the experiments that are compared and present this statistical difference.

Article Snippet: Mouse monoclonal primary antibodies used were for cyclin D1 (1:1000), BCL-2 (1:500), β-actin (1:2000), and GAPDH (1:2000), with these last two used as a cytoplasmic housekeeping protein (Nrf2, NF-κB, active caspase-3, histone H1, cyclin D1, GAPDH and secondary antibodies were purchased from Merck Millipore, Burlington, MA, USA; BCL-2 from Thermo Fischer Scientific, Rockford, IL, USA; and β-actin from Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Western Blot