Af617

Equal concentration of exosomes and cell lysates were loaded in mini-protean TGX precast gel (Bio-Rad) after addition of sample buffer and a heating step. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare). Blocking of the membranes was performed utilizing 3% ECL prime blocking agent (Amersham, GE Healthcare) for 1 h at room temperature. The membranes were exposed to an appropriate amount of primary antibodies such as CD40L (AF617, R&D Systems), 4-1BBL (ab126274, Abcam), GAPDH (mAbcam 9484, Abcam), CD63 (MX-49.129.5, Santa Cruz), or CD81 (5A6, Santa Cruz) and incubated at 4°C overnight while rotating. After a washing step, peroxidase-conjugated secondary antibodies such as mouse-immunoglobulin Gκ (IgGκ; BP-HRP, Santa Cruz), horseradish peroxidase (HRP)-conjugated anti-goat IgG (HAF109, R&S Systems), or HRP-conjugated anti-rabbit IgG (ab97051, Abcam) were added to the membranes, which were then washed with PBS/0.05% Tween 20 before development by enhanced chemiluminescence (ECL) prime western blotting detection reagents (Amersham, GE Healthcare). The protein bands were visualized by a ChemiDoc touch imaging system (Bio-Rad).

Negative contrast staining was used to evaluate the morphology and purity of the exosomes. Immunoelectron microscopy (IEM) was performed to reveal the expression of TMZ-CD40L and 4-1BBL on exosomes. Briefly, exosomes were fixed with 2% paraformaldehyde (PFA) and adsorbed to gold formvar/carbon-coated grids (Ted Pella). Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each). Finally, exosomes were observed with an electron microscope (Tecnai Biotwin) at 80 kV. For negative contrast staining, exosomes were treated with 1% glutaraldehyde and a drop of uranyl-oxalate solution, followed with 4% uranyl acetate and 2% methylcellulose after fixation and adsorption to the grids.