Purified EVs or cell lysates were added to reducing buffer in 1 : 1 ratio and boiled for 10 min at 95°C. Samples were loaded in a 4-20% 1.5 mm Bio-Rad precast gel and allowed to migrate at 100 V. Proteins were transferred to a nitrocellulose membrane in a transfer chamber at 45 mA overnight. The membrane was blocked in 5% nonfat dry milk prepared in 0.2% Tween-20 and 1× TBS for 30-45 min at RT. Primary antibodies CD9 (1 : 250), Hsp70 (1 : 250), NF-κB (1 : 250), and IRF-8 (1 : 250) (DSHB, Iowa City, IA, USA) were used in probing the membranes overnight at 4°C. Nitrocellulose blots were washed three times in wash buffer (0.2% Tween-20 in 1× TBS) for 10 min and incubated with HRP-conjugated secondary antibody (1 : 500-1 : 2000) diluted in blocking solution for 1 h at RT with gentle shaking. The membrane was washed three times in wash buffer for 10 min and developed using SuperSignal West Femto Maximum Sensitivity Substrate (vendor). The signal was detected using Bio-Rad ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA) [24 (link)].
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