Odyssey classic infrared imaging system

Two different labeling dyes were used to monitor EV uptake: a green RNA dye (Syto RNASelect Green; Invitrogen, Grand Island, NY), and a near-infrared lipid dye (DiOC18(7) or DiR; Invitrogen). For Syto RNASelect Green staining, 100 µg EV in 100 µL TBS were incubated with the dye at a final concentration of 10 µM for 30min at 37°C. For DiR staining, 62.5 µg EV in 100 µL TBS were incubated with the dye at a final concentration of 100 ng per microgram of EV for 1 h at 37°C. After incubation, unincorporated dye was removed by gel filtration using PBS-hydrated Exosome Spin Columns (3kDa MWCO, Invitrogen) (See Supplementary Fig. S7).
We also checked the stability of DiR staining of EV over 24h (Fig. S8). DiR-stained EV were prepared as above, and incubated for 24h at 37°C. We then repeated the gel filtration procedure to remove any dye that may have become unincorporated from EV during the 24h incubation. For comparison, we prepared freshly stained EV as above, and immediately performed a second gel filtration to account for EV losses in the Exosome Spin Columns. Stained EV from these samples were diluted 1:2, and 60 µL loaded in each well of a black-walled 96-well plate. Fluorescence intensity analysis of samples was performed in triplicate using the Odyssey Classic infrared imaging system (Li-Cor, Lincoln, NE) at the 800nm channel.

Cell lysates were prepared for immunoblot analysis as previously described (Silva et al., 2014 (link); Silva and McMahon, 2014 (link)). Membranes were probed with the primary antibodies as described in Supplementary Table S3. Antigen-antibody complexes were detected using fluorescent goat anti-Rabbit or anti-Mouse IRDye 800 or goat anti-Mouse IRDye 680 secondary antibodies (LI-COR Biosciences) and visualized using a LI-COR Odyssey Classic infrared imaging system. Immunoblot data was analyzed using the Odyssey application software v3.0.30 software (LI-COR Biosciences) (Silva and McMahon, 2014 (link)).

Nuclei were isolated using a commercial nuclear extract kit (Active Motif, Carlsbad, CA). Protein concentrations were measured at 750nm using bovine serum albumin as the standard (DC™ Protein Assay Kit, Bio-Rad, Hercules, CA). Equal nuclear protein volumes were added to a TransAM™ assay plate (Active Motif) coated with immobilized oligonucleotide containing the GR consensus binding site (5′-GGTACAnnnTGTTCT-3′). GR activity was assessed per the manufacturer’s instructions with an optical density of 450 nm. SDS-PAGE was performed using 30 μg of nuclear lysate loaded onto a precast gradient (4-20%) gel (Bio-Rad). Proteins were transferred onto a nitrocellulose membrane (Bio-Rad) and incubated with human GR antibody raised in rabbit (Santa Cruz) and IRDye 800CW Donkey anti-Rabbit IgG and IRDye 680RD Donkey anti-Goat IgG (Li-Cor Biosciences, Lincoln, NE). Membranes were visualized on an Odyssey Classic Infrared Imaging System and densitometry performed using Image Studio Lite version 4.0 (Li-Cor Biosciences).