Journal: Journal of Neuroinflammation

Article Title: Anti-tat Hutat2:Fc mediated protection against tat-induced neurotoxicity and HIV-1 replication in human monocyte-derived macrophages

doi: 10.1186/s12974-014-0195-2

Figure Lengend Snippet: Evaluation of the biological binding function of Hutat2:Fc and protective effects of Hutat2:Fc against HIV-1 Tat 86 -mediated toxicity in HTB-11 cells. (A) Specific binding of Hutat2:Fc to HIV-1 Tat. HIV-1 Tat 86 (Clade B) loaded nitrocellular membranes (NCM) were incubated with cell culture supernatants collected from HR-Hutat2-transduced HTB-11 (HTB-Hutat2), U937 (U937-Hutat2), or hMDM (hMDM-Hutat2) at 4°C overnight followed by incubation with rabbit anti-human IgG (H+L) and goat anti-rabbit IgG HRP conjugated antibodies. Specific binding was visualized by the color deposition on the NCM. The Tat 86 -loaded membrane incubated with rabbit anti-Tat serum served as a positive control (Pos Ctl) while incubated with cell culture supernatant from HR-A3H5 transduced HTB-11 served as a negative control (HTB-A3H5). The NCM loaded with Tat dilution buffer was used as a blank control (BLK Ctl). (B) Functional antagonization of Hutat2:Fc against HIV-1 Tat 86 -induced toxicity in HTB-11 cells by an MTT assay. The OD 570 value of untreated HTB-11 cells was arbitrarily defined as 100% cell viability. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. The cell viability was significantly higher for the cells treated with the conditioned mediums from transduced cells releasing Hutat:Fc when compared to the cultures that received Tat 86 (500 nM) alone (* P <0.01 for HTB-Hutat2 medium; # P <0.05 for U937-Hutat2 medium, and hMDM-Hutat2 medium). (C) Protection of HR-Hutat2 transduction against Tat 86 -induced toxicity by an MTT assay. No significant difference of cell viability was detected between normal and vector HR-Hutat2 transduced HTB-11 cells (HTB-Hutat2) ( P >0.05). However, the cell viability of HTB-11 transduced with the vector HR-Hutat2 was significantly higher than that of HTB-A3H5 in the presence of HIV-1 Tat 86 (500 nM) (* P <0.01). All experiments were performed in quadruplicate. Error bars denote the s.e.m.

Article Snippet: After washing three times with TBST, the plate was incubated with diluted Hutat2:Fc containing supernatant samples for 1 hour and then incubated with a goat anti-human IgG Fc biotin-conjugated detection antibody (Rockland) for 1 hour.

Techniques: Binding Assay, Incubation, Cell Culture, Membrane, Positive Control, Negative Control, Control, Functional Assay, MTT Assay, Transduction, Plasmid Preparation