Anti b7 h6

Cells were homogenized briefly in 10 volumes of lysis buffer containing (in mM) 20 Tris–HCl (pH 7.4), 150 NaCl, 2.5 EDTA, 50 NaF, 0.1 Na₄P₂O₇, 1 Na₃VO₄, 1 PMSF, 1 DTT, 0.02% (v/v) protease cocktail (Sigma Aldrich, St. Louis, MO, USA), 1% (v/v) Triton X-100 and 10% (v/v) glycerol. The homogenates were centrifuged twice at 20,000×g at 4 °C for 15 min, and the supernatants were retained as total protein. Protein concentrations were determined by the BCA method. Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, MA, USA). Western blot analysis was performed under standard conditions with specific anti-B7-H6 (1:2000; Abcam, MA, USA), anti-C-myc (1:1500, Abcam, MA, USA), anti-C-fos (1:2000, Cell Signaling Technology, MA, USA), anti-cyclin D1 (1:2000, Cell Signaling Technology, MA, USA), and anti-GAPDH (1:4000, Sigma, St. Louis, MO, USA) antibodies and HRP-labeled goat anti-mouse/rabbit secondary antibody (1:6000, Sigma Aldrich, St. Louis, MO, USA). The immunoreaction was visualized using an enhanced chemiluminescence detection kit (Thermo Fisher, MA, USA) and exposure to X-ray film, and band densities were quantified by densitometry with a video documentation system (Gel Doc 2000, Bio-Rad).

Cells were lysed with 1× radioimmunoprecipitation assay (RIPA) buffer, and EVs, resuspended in 1× PBS, were lysed in 5× RIPA buffer; the protein concentration was determined by Pierce^TM BCA protein assay (Thermo Fisher Scientific). A 4× SDS sample buffer was added, and samples were run on a 10% SDS protein gel. Proteins were transferred to a nitrocellulose membrane (GE Healthcare, Freiburg, Germany), and membranes were blocked with 5% (w/v) milk powder (Carl Roth) in Tris-phosphate-buffered saline supplemented with 0.05% Tween (TBS-T) for 1 h. Subsequently, membranes were probed using the following antibodies: anti-Calnexin (clone AF18, Santa Cruz Biotechnology, Heidelberg, Germany), anti-Flotillin-1 (clone 18, BD Biosciences), anti-GAPDH (G9545, Sigma-Aldrich, Munich, Germany), anti-BAG6, and anti-B7-H6 (ab121794, Abcam, Cambridge, UK). Detection was performed with horseradish peroxidase-conjugated secondary antibodies (DAKO, Hamburg, Germany) using Amersham ECL Plus (GE Healthcare).