Animage quant las4000

To prepare brain homogenates, tissue was removed from the frontal lobe,
lysed in strong lysis buffer (detailed above) with proteinase inhibitor,
homogenized by pipette, and centrifuged at 10,000 × g for 10 minutes to
discard whole cells and intact cellular debris. For confirmation of EV proteins
in purified lysates, 5X Laemmli sample buffer [10% SDS, 250 mM Tris pH 6.8, 1
mg/mL bromophenol blue, 0.5 M DTT, 50% glycerol, 5% BME] was added to EV
isolates for a final concentration of 1X. For immunoblot analysis of CD63, DTT
and BME were omitted from lysis and sample buffers to allow the protein to run
in non-reducing conditions. Gel electrophoresis and western blotting was
performed as previously described (Hurwitz et
al., 2017b). Equal volume of gradient lysates were loaded into an
SDS-PAGE gel. Blots were probed with the following antibodies: Alix (Q-19; Santa
Cruz Biotechnology), HSC70 (B-6; Santa Cruz), TSG101 (C-2; Santa Cruz), CD63
(TS63; Abcam), Rab8a (63-BJ ; Santa Cruz), and CD81 (H-121 ;Santa Cruz), rabbit
anti-mouse IgG (Genetex, 26728), rabbit anti-goat IgG (Genetex, 26741), goat
anti-rabbit IgG (Fab fragment) (Genetex, 27171). Blots were imaged using an
Image Quant LAS4000 (General Electric) and processed with ImageQuant TL v8.1.0.0
software, Adobe Photoshop CS6 and CorelDraw Graphic Suite X5.

DCIS cells starved overnight. Next day, 20% DCIS-Δ4 cells were
added to the DCIS cells for 0 or 30min. Cell lysates were prepared and blotted
on the Human Phospho-kinase array (R&D, #ARC003C) according to the
manufacturer’s protocol. Membranes were imaged using an
ImageQuant™ LAS 4000 biomolecular imager (GE) and signal intensity was
quantified using ImageJ, RRID:SCR_003070.