anti-human endogenous retrovirus type k (herv k) envelope protein. igg fraction  (AMS Biotechnology)

Journal: Journal of Carcinogenesis

Article Title: Human endogenous retroviral K element encodes fusogenic activity in melanoma cells

doi: 10.4103/1477-3163.109032

Figure Lengend Snippet: Antibody neutralization assay. 10 4 each of A375 cells stably expressing pS-puro-scrambled and pEYFP-N3-neo were added per well in 96 well plate. Different dilutions of two K type human endogenous retrovirus envelope monoclonal antibodies HERM-1811-5 and HERM-1821-5 were added to culture medium. PBS was used as control. Cells were cultured for 48 h, followed by selection of pS-puro-scrambled-pEYFP-N3 fused cells using puromycin and G418 double selection for 48 h. The numbers of blue-green double color cells (fused cells) as shown in 3a upper left were calculated and compared between antibody treated and control

Article Snippet: [ ] To examine whether ENV plays a role in intercellular fusion, we performed an immunoneutralization assay using commercially available HERV-K ENV monoclonal antibodies HERM-1811-5 and HERM-1821-5 (Austral Biologicals, San Ramon, CA).

Techniques: Neutralization, Stable Transfection, Expressing, Cell Culture, Selection

Journal: Retrovirology

Article Title: Trans-activation, post-transcriptional maturation, and induction of antibodies to HERV-K (HML-2) envelope transmembrane protein in HIV-1 infection

doi: 10.1186/1742-4690-11-10

Figure Lengend Snippet: Evidence of trans-activation and post-transcriptional modification of HERV-K (HML-2) Env TM. A) HERV-K (HML-2) Env mRNA expression was detected using primers designed to bind the SU domain or TM domain (SU primers or TM primers respectively) at 0,1 and 2 days post infection. Copies of HERV-K (HML-2) Env detected by TM primers (plain line) increased by the time of infection compared to the copies detected by SU primers (dashed line). The graph is a representative experiment of 3 individual independent experiments. Copy number was determined as described previously, and β-Actin was used as reference gene . B) The graph represents cumulative data from 3 independent experiments and shows the ratio of copies of HERV-K (HML-2) Env detected by TM primers /copies detected by SU primers from HIV-1 LAI infected PBMCs 2 days post-infection. Similar data were obtained using primary isolates (91US_4/R5 tropic and BK132/X4 tropic). C) Nested PCR. The figure shows the amplicons obtained after the second round of PCR (around 500pb). TM mRNA is over-expressed during the peak of viremia at d42. Antiviral treatment induced the transcription of both SU and TM mRNA at a similar level. HIV neg : HIV-1 seronegative low risk donor; OP-1830: HIV-1 seroconverter patient (d0: before infection; d12, d42, d76: after infection without treatment, d104: after treatment); water: the non-template well. (D) Assumed precursor proteins at 75 to 90 kDa and TM subunits at 32 to 38 kDa are visible. Hela-T4 cells are infected by HIV-1LAI in presence (4) or not (3) of 10 μl/ml of tunicamycin. HERK-Env transfected cells (2) were used as positive controls for HERV-K TM expression. Uninfected-untransfected cells were used as control for endogenous HERV-K basal expression (1). (E) Representative images of HERV-K TM extracellular expression on PBMCs. HERM-1811-5 (anti-TM) mouse monoclonal antibody and goat anti-mouse Alexa555 (red) were used to detect extracellular expression of HERV-K (HML-2) TM.

Article Snippet: PVDF (Biorad) membranes were blocked 1 h at RT in PBS/0.05%-Tween 20/10%-non fat dry milk and incubated with mouse monoclonal anti-HERV-K TM HERM-1811-5 (Austral Biologicals) in PBS/0.05%-Tween 20/5%-nonfat dry milk at 1/1000 over-night at 4°C.

Techniques: Activation Assay, Modification, Expressing, Infection, Nested PCR, Transfection