Journal: Journal of visualized experiments : JoVE

Article Title: Quantification of protein phosphorylation using single molecule pull-down

doi: 10.3791/63665

Figure Lengend Snippet: Table of Materials

Article Snippet: Mix-n-Stain™ CF® Dye Antibody Labeling Kits , Biotium , 92245 , Suggested conjugation kit.

Techniques: Electron Microscopy, Lysis, Recombinant, Bicinchoninic Acid Protein Assay, Convection, Protease Inhibitor, Plasmid Preparation, Antibody Labeling, Conjugation Assay, Binding Assay, Microscopy, Cell Counting, Transferring, Staining, Software

Journal: AIDS (London, England)

Article Title: Activation of the signal transducer and activator of transcription 1 signaling pathway in thymocytes from HIV-1-infected human thymus

doi: 10.1097/01.aids.0000060415.18106.1a

Figure Lengend Snippet: (a) Quantitative measurement of differential gene expression. RNA from six different yeast-specific genes was mixed with the HIV or mock-infected RNA samples in the microarray assay. RNA from each yeast gene was added at a known ratio (3, 10 or 25) in the Cy3 (HIV) or Cy5 (mock) sample (Cy3/Cy5 added). The six yeast genes were arrayed on the slides in quadruplets. Positive values indicate threefold, 10-fold or 25-fold more RNA were used in the Cy3 (HIV) sample. Negative values (−3, −10 or −25) indicate that threefold, 10-fold or 25-fold more RNA were included in the Cy5 (mock) sample. An equal amount of RNA in Cy3 and Cy5 samples is indicated as 1 (+1 or −1). The X axis shows the added ratios and the Y axis indicates Cy3/Cy5 readout data from the assay. Error bars indicate standard deviations. (b) RNA sample and reaction control. RNA samples from HIVJD (Cy3 labeled) or mock (Cy5 labeled) infected severe combined immunodeficient (SCID)-hu Thy/Liv mice were analysed using the microarray assay. Relative expression levels of 23 000 Mr highly basic protein (HBP), ribosomal subunit S9 (S9 rRNA) and α-tubulin were quantified in quadruplets. Three sets of human genes known to express in multiple tissues (complex target 1–3) were also included as controls. Positive values (Cy3 signal/Cy5 signal) indicate more RNA in the Cy3 (HIV) sample, thus induction of expression by HIV. Negative values (Cy5 signal/Cy3 signal) indicate more RNA in the Cy5 (mock) sample, thus suppressed expression by HIV. Error bars indicate standard deviations from the quadruplet arrays. The relative expression of three T cell markers (TCRβ, CD8 and Thy-1) from the microarray assay was also shown. Comparison of HIV (JD or NL4-3) and mock infected samples from different donor tissues showed similar results. (c) Alteration of gene expression in SCID-hu Thy/Liv mice after HIV-1 infection. A small number (< 0.2%) of genes were induced (> threefold) in HIV-1-infected Thy/Liv organs over mock infected samples. The majority of genes were in the +2.5 to −2.5 range (99.5%). Error bars indicate standard deviations from three experiments (two with JD and one with NL4-3) with different donor tissues.

Article Snippet: Monoclonal antibodies reactive with human CD4 and CD8 cells were purchased from Becton Dickinson (San Jose, CA, USA).

Techniques: Gene Expression, Infection, Microarray, Control, Labeling, Expressing, Comparison