Journal: PLoS Pathogens
Article Title: Using the TAP Component of the Antigen-Processing Machinery as a Molecular Adjuvant
doi: 10.1371/journal.ppat.0010036
Figure Lengend Snippet: (A) Human TAP1 protein expression in mouse splenocytes was determined by Western blot. The mice infected with VV-PJS-5 were used as negative controls for human TAP1 expression. (B) The expression of human TAP1 and human TAP2 was detected by RT-PCR 24 h after the mice were infected with VV-hTAP1,2. The mice infected with VV-PJS-5 were negative for human TAP1 and TAP2. (C) Immunofluorescence visualized with confocal microscopy identified human TAP1 expression in antigen-presenting splenocytes isolated from mice 24 h after infection with VV-hTAP1,2. The mice infected with VV-PJS-5 were used as negative controls for human TAP1 expression (green fluorescence) (I). Cell-surface markers (red fluorescence) identified cell types. Representative images show the following cell types: (I) B cell from a mouse infected with VV-PJS-5 (negative control); (II) B cell that is positive for human TAP1; (III) macrophage that is positive for human TAP1; and (IV) DC that is positive for human TAP1. (D) ATP-dependent TAP activity was measured in splenocytes taken 24 h after the mice were infected with VV-hTAP1,2 or VV-PJS-5 (negative control). Active transport activity was measured in the presence or absence of ATP by a peptide-transport assay that determined the translocation of radioactive peptides from the cytosol into the ER. Normal uninfected mice, uninfected TAP −/− mice, and mice infected with VV-PJS-5 were used as negative controls when assessing the effect of VV-hTAP1,2 infections on peptide-transport activity. The bars represent the mean value ± standard error of the mean of triplicate measurements. The data are representative of the experiment performed in duplicate.
Article Snippet: Splenocytes were double labeled with rabbit anti-human TAP1 antiserum (Stressgen Biotechnologies) and one of the following cell-surface markers: rat anti-mouse B220 (B cell marker, BD Biosciences Pharmingen), rat anti-mouse MAC-1 (macrophage marker, BD Biosciences Pharmingen), or rat anti-mouse NLDC-145 antibodies (DC marker) (gift from Ralph Steinman, the Rockefeller University, New York, New York, United States).
Techniques: Expressing, Western Blot, Infection, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Confocal Microscopy, Isolation, Fluorescence, Negative Control, Activity Assay, Transport Assay, Translocation Assay
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