Lamp2b

Total proteins were detached by 10% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane. After that, the membrane was incubated with primary antibody against BCL6 (1:500, cat. no. #5650, Cell Signaling Technology), integrin αv (1:1000, cat. no. ab179475, Abcam), integrin β3 (1:500, cat. no. ab119992, Abcam), CD63 (1:500, cat. no. AF5117, Affbiotech), CD81 (1:500, cat. no. 27855-1-AP, Proteintech), TSG101 (1:1000, cat. no. 28283-1-AP, Proteintech), HSP70 (1:1000, cat. no. ab2787, Abcam), calnexin (1:1000, cat. no. ab133615, Abcam), Lamp2b (1:500, cat. no. ab18529, Abcam) and β-actin (1:10000, cat. no. TDY051, Bejing TDY Biotech Co., LTD.) at 4°C overnight. After incubating with the HRP-labeled goat anti-rabbit secondary antibody for 1 h at room temperature, protein bands were visualized by the ECL reagents.

EVs were lysed with 40 µL of RIPA buffer supplemented with protease inhibitor. Then, 10 µg of total protein were prepared for Western blot analysis. Twenty micrograms of EVs protein were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PVDF membrane (Millipore). Membranes were blocked with 5% skim milk for 1 h at room temperature and were immunostained with primary antibody against TSG101 (Abcam) or LAMP2b (Abcam), at a dilution of 1:1000 overnight at 4 °C, and corresponding secondary antibody 1 h at room temperature. The Clarity and Clarity Max ECL Substrates (Bio-Rad) were applied for detecting specific bands on membranes.

Cells were lysed on ice for 30 min with RIPA buffer, protease inhibitor cocktail ± phosphatase inhibitor cocktail. Cell lysate proteins (80 μg) were resolved on SDS-PAGE and transferred to nitrocellulose for western blots. Blots were quantitated by densitometry using ImageJ (NIH, Bethesda, MD, United States) and normalized to cellular Actin. Antibodies against LAMP-2A (Cat #ab18528), LAMP-2B (Cat #ab18529), HSP90 (Cat #ab13494), and cathepsin A (Cat #ab79590) were from Abcam (Cambridge, MA, United States). Chk1 (Cat #2360), phospho-Chk1 (Ser345) (Cat #2341), IκBα (Cat #4814), phospho-IκBα (Ser32/36) (Cat #9246), LC3B (Cat #2775), and histone H3 (Cat #3638) were from Cell Signaling Technology (Danvers, MA, United States). LAMP-2 (Cat #H4B4-c) was from DSHB (Iowa City, IA, United States) and HSC70 (Cat #ADI-SPA-815) from Enzo Life Sciences (Farmingdale, NY, United States). Anti-Myc Tag (Cat #05-724) and cathepsin D (Cat # IM03) were from EMD Millipore (Billerica, MA, United States). Cathepsin B (Cat # sc-13985), p53 (Cat # sc-126), and p21 (Cat # sc-756) were from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Actin (Cat # MS-1295-P0) was from Thermo Fisher Scientific.