Figure 1
From: Development of a quantitative method to measure EV uptake
Characterization of DSP constructs. (A) Subcellular localization of the different DSP constructs. SUM159 cells were transiently transfected with the indicated DSP constructs and stained with α-GFP rabbit polyclonal Ab. (B) Dual Split Proteins reassociation detected by fluorescence microscopy. SUM159 cells were co-transfected with DSP1 + DSP2, DSP1-CD9 + DSP2 or DSP1-CD63 + DSP2 and EGFP signal was observed by fluorescence microscopy 48 h after transfection. (C) Staining of DSP contructs with anti-tetraspanin antibodies. SUM159 cells were co-transfected with DSP1-CD9 + DSP2 or DSP1-CD63 + DSP2 and stained with VJ1/20 anti-CD9 or Tea3/10 anti-CD63 mAbs, respectively. EGFP signal and anti-tetraspanin stainings were observed by fluorescence microscopy 48 h after transfection. All samples were co-stained with DAPI. Images were acquired in a wide-field epifluorescence microscope. Bars = 20 μm. (D) SUM159 cell lines that stably express each DSP construct were obtained by transduction with a pLVX lentiviral vector, lysed and subjected to SDS-PAGE. (E) Lysates from EVs isolated by ultracentrifugation at 100000 g from SUM159 expressing DSP1-CD9 or DSP1-CD9/DSP2 conditioned media. Membranes were blotted with α-GFP rabbit polyclonal antibody or α-CD9 (VJ1/20) mouse mAb. Positions of molecular weight markers and the different specific bands are indicated.
