pbabe puro vector  (Addgene inc)

Journal: eLife

Article Title: Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across five fly Parkinsonism models

doi: 10.7554/eLife.98348

Figure Lengend Snippet:

Article Snippet: At day 50, DAN were transduced with a lentiviral vector expressing mRuby2-T2A-GCaMP6f (Addgene plasmid # 197595).

Techniques: Knock-Out, Knock-In, Control, Mutagenesis, Transfection, Construct, Plasmid Preparation, Expressing, Imaging, Recombinant, Sequencing, Multiplex Assay, Software

Journal: Viruses

Article Title: HTLV-1 p13 Protein Hijacks Macrophage Polarization and Promotes T-Cell Recruitment

doi: 10.3390/v17040471

Figure Lengend Snippet: P13 targets mitochondria in monocytic cells. ( A ) Schematic representation of p13-HA-GFP viral protein. The p13 protein fused to HA and GFP carboxy terminal tags was cloned in a retroviral vector pBABE. The N-terminal region of p13 has a mitochondrial targeting sequence (MTS) from amino acids 21 to 35. ( B ) Immunoblot for HA expression from total cellular extracts of THP-1 cells transduced with retrovirus expressing p13-HA-GFP. Cells transduced with empty retrovirus were used as control (Ctrl). β-Actin expression was used as a loading control. ( C ) Stable cell lines THP-Ctrl or THP-p13 were adhered to glass slides by cytospin and stained with an antibody to complex IV (COXIV). Scale bar: 10 µm. ( D ) Proliferation assay of THP-Ctrl or THP-p13. Cells were stained with CellTrace™ Far Red, and MFI was measured by flow cytometry every 24 h for three days. The results of three independent experiments were graphed; Ctrl is labeled in blue, and p13-expressing cells in red. Statistical significance was verified with a Student’s t -test. No statistical significance was noted between p13-expressing cells and Ctrl. ( E ) THP-Ctrl and THP-p13 cells following the treatment with increased concentration of Staurosporine (0.01, 0.1, 1 μM). Cells were stained with Live/Dead Fixable Blue dye (ThermoFisher Scientific) to measure the percentage of live cells every 24 h for three days by flow cytometry. The results of three independent experiments were graphed; Ctrl and p13 cells are labeled in blue and red, respectively. Statistical significance was verified with a Student’s t -test. No statistical significance was noted between p13-expressing cells and Ctrl. ( F ) Seahorse extracellular flux analysis measured the oxygen consumption rate in THP-p13 or THP-Ctrl cells. A representative rate of spare respiratory capacity is shown. Different mitochondrial parameters are as follows: basal respiration (yellow), ATP-linked respiration (green), proton leak (magenta), maximal respiratory capacity (blue), and non-mitochondrial respiration (gray). ( G ) The maximal respiratory capacity rate was graphed for THP-Ctrl (blue) and THP-p13 (red). Statistical significance was verified with a Student’s t -test and reported in the figure. The p -values are summarized with asterisks, *** ( p ≤ 0.001). ( H ) Seahorse extracellular flux analysis measured the oxygen consumption rate in p13-expressing and control THP cells.

Article Snippet: The p13-HA-GFP construct encodes the mammalian codon-optimized p13 (with a modification in amino acid number 13 from glutamic acid [E] to glycine [G]) in-frame with an HA and GFP tag in a retroviral pBABE-puro vector. pBABE-puro was kindly provided by Dr. Hartmut Land, Dr. Jay Morgenstern, and Dr. Robert Weinberg (Addgene plasmid # 1764; https://www.addgene.org/1764/ [accessed on 24 March 2025]; RRID: Addgene_1764) [ ].

Techniques: Clone Assay, Retroviral, Plasmid Preparation, Sequencing, Western Blot, Expressing, Transduction, Control, Stable Transfection, Staining, Proliferation Assay, Flow Cytometry, Labeling, Concentration Assay