Grp78 bip

Exosome and cell lysates were separated by SDS-PAGE using precast, 4–15% gradient gels and transferred to PVDF membranes. Membranes were blocked for 2 h using 5% nonfat dry milk in Tris-buffered saline (TBS) with Tween-20 (TBST; 138 mM NaCl, 2.7 mM KCl, 50 mM Tris, pH 8.0, 0.05% Tween-20). Membranes were then incubated overnight in blocking solution containing antibodies obtained from commercial sources (CD9 [clone HI9a, BioLegend]; CD63 [clone MX-49.129.5, Santa Cruz Biotechnology], CD81 [555675, BD Pharmingen], E-cadherin [20874-1-AP, Thermo Fisher]; N-cadherin [MA1-2002, Thermo Fisher]; GRP78/BiP [PA1-014A, Thermo Fisher]; calreticulin [MA5-32131, Thermo Fisher]; ERGIC-3 [clone B5, sc-398778, Santa Cruz Biotechnology]; GM130 [PA1-077, Thermo Fisher]; HSP60 [ab190828, Abcam]; HSP90 [sc-13119; Santa Cruz Biotechnology]; Lamp2 [MA1-205, Thermo Fisher]). The next day, the membranes were washed, exposed to HRP-conjugates of goat secondary antibodies (Jackson Immunoresearch), washed again, incubated with an HRP-activated chemiluminescence detection solution (Amersham), and imaged using a GE Amersham Imager 600. Images were exported as JPEG files and assembled into figures using Adobe Illustrator and Adobe Photoshop.

Cells were seeded on glass coverslips placed in 6-well plates and treated for 96 h with Dox. They were then fixed with 3.7% paraformaldehyde for 10 min, permeabilized in 100% ice-cold methanol for 10 min at −20 °C and finally blocked with DPBS supplemented with 3% bovine serum albumin (BSA) for 30 min. After each step, cells were rinsed in DPBS and incubated with primary antibodies overnight at 4 °C, following which the cells were washed in DPBS and incubated with secondary antibodies at RT for 1 h on the next day. Glass coverslips were counterstained with DAPI, mounted on glass slides and examined under a Zeiss confocal fluorescence microscope (Zeiss LSM 700). The primary antibodies used were the following: Calreticulin (D3E6) XP (Cell Signaling Technology (CST), Heidelberg, Germany, 12238; 1:250), Dyskerin (Santa Cruz Biotechnology, Dallas, TX, USA, sc-373956; 1:1000), GRP78/BiP (Thermo Fisher Scientific, Waltham, MA, USA, PA5-11418; 1:500) and LC3 (Abcam, CA, USA, ab51520; 1:3000). The secondary antibodies used were: Cy3-AffiniPure Goat Anti-Rabbit IgG (H + L) (Invitrogen, Van Allen Way Carlsbad, CA, USA, A10520; 1:500) and Alexa Fluor 488 donkey anti mouse IgG (H + L) (Invitrogen, Van Allen Way Carlsbad, CA, USA, A21202; 1:1000).