Tsg101

The right lung tissues were cut into 2-3 mm³ debris and prepared as tissue homogenate by a handheld homogenizer. The protein concentration was determined using the bicinchoninic acid method. Then, an equal volume of protein was run on 5-12% SDS PAGE electrophoresis and transferred on PVDF membranes. After being blocked by 5% BSA, the membranes were incubated with the primary antibodies to α-tubulin (Sigma, T6199), CD63 (Abcam, ab217345), CD9 (Abcam, ab92726), TSG101 (Thermo Fisher, PA5-82236), Calnexin (Immunoway, YP0041), FGF1 (CUSABIO), MAPK (CST, 8690), p-MAPK (CST, 4370), STAT1 (CST, 9172), and p-STAT1 (CST, 9167) at 4 °C overnight, and then with HRP-conjugated secondary antibodies at room temperature for 1 hour. The bands were detected by enhanced chemiluminescence detection kit (Thermo Fisher, 32106) and visualized via the DNR western blot detection system.

Equal amounts of total proteins from the EVs samples or cell lysates were separated on 10% SDS-PAGE gels and then transferred onto PVDF membranes (PerkinElmer, USA). After blocking for 2 h at room temperature with 5% skimmed milk in trimethyl benzene sulfonyl tetrazole buffer, the membranes were incubated overnight at 4 °C with gentle shaking with antibodies against CD 63 (1:2000, Thermo Fisher Scientific), GAPDH (1:2000, rabbit, Thermo Fisher Scientific), TSG101 (1:500, rabbit, Thermo Fisher Scientific), histone H3 (1:1000, rabbit, Abcam), TLR4 (2 μg/ml, rabbit, Thermo Fisher Scientific), and p-NFκB-p65 (1:1000, rabbit, Thermo Fisher Scientific). Then the membranes were incubated with secondary antibodies for 30 min at room temperature. Finally, the immunoreactive protein bands were visualized using an enhanced chemiluminescence reagent, followed by imaging on an electrophoresis gel imaging analysis system (DNR Bio-Imaging Systems, Israel).

Protein expression was examined by SDS-PAGE, as described previously [20 (link)]. Briefly, total intracellular proteins were extracted from the cells by freeze/thawing in lysis buffer containing protease inhibitors. Protein content was estimated according to Biorad protein assay (BIO-RAD). A total of 20 µg of proteins were visualized using the chemioluminescence detection system (Amersham biosciences; Little Chalfont, UK) after incubation with rabbit polyclonal primary antibodies against ANXA1 (1:10,000; Invitrogen; Carlsbad, CA, USA) and calreticulin (1:1000; Elabscience; Houston, TX, USA), with mouse monoclonal primary antibodies against TSG101 (1:1000; ThermoFisher Scientific; Waltham, MA, USA) and anti-β actin (mouse monoclonal; 1:1000; clone AC15; A5441, Sigma-Aldrich). The blots were exposed and analysed to Las4000 (GE Healthcare Life Sciences).

Primary antibodies and the respective dilutions used for Western blotting included annexin V (Abcam, Cambridge, United Kingdom; ab117439) (1:1,000), CD9 (Cell Signaling Technologies, Danvers, MA; CST 13174S) (1:1,000), CD81 (Systems Biosciences, Palo Alto, CA [1:1,000], or BD Biosciences, San Jose, CA [1:2500], or Thermo Fisher Scientific, Waltham, MA [MA5-13548] [1:100]), flotillin-1 (CST 18634S) (1:1,000), calnexin (Santa Cruz Biotechnology, Inc., Dallas, TX; sc-11397) (1:200), cytochrome c (BD Pharmingen 556433) (1:500), TSG101 (Thermo PA5-31260) (1:500), and PAB597 (purified) (1:2,000). PAB597 is a monoclonal antibody against VP1 (46 (link)). Secondary antibodies used for Western blotting included anti-Mus horseradish peroxidase (HRP) (Thermo A28177) and anti-rabbit HRP (Thermo A27036), both used at 1:10,000. Anti-rabbit 680, anti-mouse 680, anti-rabbit 800, and anti-mouse 800 (Li-Cor, Lincoln, NE) were all used at 1:5,000.

Proteins extracted from cells and EVs were examined by Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE). Protein content was estimated according to the Biorad protein assay (BIO-RAD, Hercules, CA, USA), as previously described [17 (link)]. We have analyzed primary antibodies against rabbit polyclonal ANXA1 (1:10,000; Invitrogen; Carlsbad, CA, USA), calreticulin (1:1000; Elabscience; Houston, TX, USA), and mouse monoclonal TSG101 (1:1000; ThermoFisher Scientific; Waltham, MA, USA), CD81 (1:200; Becton Dickinson Labware, Franklin Lakes, NJ, USA), CD63 (1:200; Biolegend; San Diego, CA, USA), and GAPDH (mouse monoclonal, 1:1000; Santa Cruz Biotechnologies, Dallas, TX, USA). The blots were exposed to Las4000 (GE Healthcare Life Sciences; Little Chalfont, UK).

sEV isolated from plasma, immunocaptured sEV or cell lysates (10 ug protein) were separated on 7–15% SDS/PAGE gels and transferred onto PVDF membrane (Millipore, Billerica, MA, USA) for western blot analysis. Membranes were incubated overnight at 4°C with antibodies specific for ALIX (PDCD6IP) (1:500, #2171S, Cell Signaling), Gelsolin (1:500, #MA5‐34684, Thermo Fisher Scientific), Contactin‐1 (1:250, #MAB9041, R&D Systems), and TSG101 (1:500, PA5‐31260, Thermo Fisher). Next, the HRP‐conjugated secondary antibody (1:10,000, Pierce, Thermo Fisher) was added for 1 h at room temperature (RT) and blots were developed with ECL detection reagents (GE Healthcare Biosciences, Pittsburgh, PA, USA). The intensities of the bands on exposed films were quantified using Image J software (NIH, USA).

Proteins extracted from cells were examined by SDS-PAGE. Protein content was estimated according to Bradford assay (BIO-RAD, Hercules, CA, USA), as previously described [23 (link)]. We have analyzed primary antibodies against pPKCα (phospho-Protein Kinase C alpha; rabbit polyclonal; 1:500; Thermo Fisher Scientific, Waltham, MA, USA), ERK (rabbit monoclonal; 1:1000; Cell Signaling Technology, Danvers, MA, USA), pERK (rabbit monoclonal; 1:1000; (phospho-Thr202/Tyr204; Cell Signaling Technology, Danvers, MA, USA), calreticulin (rabbit polyclonal; 1:1000; Elabscience; Houston, TX, USA), TSG101 (tumor susceptibility gene 101; mouse monoclonal; 1:1000; ThermoFisher Scientific; Waltham, MA, USA), CD81 (mouse monoclonal; 1:500; BD Pharmingen, Franklin Lakes, NJ, USA), CD63 (mouse monoclonal; 1:500; BioLegend; San Diego, CA, USA); pSer27-ANXA1 (rabbit polyclonal; 1:500, homemade, see [24 (link)]), GAPDH (glyceraldehyde 3-phosphate dehydrogenase; mouse monoclonal; 1:1000; Santa Cruz Biotechnologies; Dallas, TX, USA) β-actin (mouse monoclonal; 1:1000; Santa Cruz Biotechnologies; Dallas, TX, USA). The blots were exposed to Las4000 (GE Healthcare Life Sciences; Little Chalfont, UK) and the relative band intensities were determined using ImageQuant software (GE Healthcare Life Sciences; Little Chalfont, UK).