Journal: PLoS ONE

Article Title: Design and Evaluation of Optimized Artificial HIV-1 Poly-T Cell-Epitope Immunogens

doi: 10.1371/journal.pone.0116412

Figure Lengend Snippet: (A) 2.5% agarose gel electrophoresis of RT-PCR products derived from isolated mRNA after 293T cells transfection with obtained plasmids. Lanes 1–3, PCR fragments of 435 bp correspond to P1, P2, and P3 plasmids; Lanes 4–6, PCR fragments of 624 bp correspond to P1, P2, and P3 plasmids; K, negative control sample (total mRNA extracted from transfected cells); M, 100bp DNA Ladder (SibEnzyme, Russia). (B) Western blot analysis of expression products of genes encoding TCI-N, TCI-N2, and TCI-N3 proteins in transfected 293T cells. Lanes P1, P2, P3 are lysates of 293T cell transfected with P1, P2, P3, respectively; lane К represents lysates of 293T cells transfected with vector plasmid pcDNA3.1. The membrane containing the cell-associated fractions was also probed with аnti-beta аctin antibody to control for equal loading of the samples. (C) Flow cytometry analyses of expression products of target genes after transfection of 293T cells with P1, P2, and P3 plasmids. Histogram overlays of target proteins stained with FITC-labeled 29F2 monoclonal antibodies (green peak, TCI-N; blue peak, TCI-N2; and yellow peak, TCI-N3 polyepitope protein expression). Negative control (read peak) represents 293T cells transfected with vector plasmid pcDNA3.1.

Article Snippet: 293T (human embryonic kidney cell line) cells were cultured in RPMI (Biolot, Russia) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL, Rockville, MD).

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Isolation, Transfection, Negative Control, Western Blot, Expressing, Plasmid Preparation, Membrane, Control, Flow Cytometry, Staining, Labeling, Bioprocessing