Ripa buffer

Cell pellets were resuspended in ice-cold RIPA buffer (Thermo Fisher) supplemented with 40 U/ml rRNAseIn Plus (Promega), 0.5 mM DTT, 5 mM EDTA and 1 tablet/25 ml mini cOmplete protease inhibitor cocktail (Roche). Cells were sonicated three times at 8 W: 20 s on, 2 min off, in an ice bath. Lysates were spun down and Protein G Dynabeads (Invitrogen) were prepared to pre-clear the lysates by washing with RIPA buffer twice. Pre-clearing was performed on the nutator for 30 min at 4°C. After this, pre-cleared lysates were incubated with 5 μg antibody for 2 h at 4°C on nutator (anti-PABPC, ab21060 Abcam; anti-PABPN, [EP3000Y] ab75855 Abcam; isotype control anti-rabbit IgG monoclonal ab172730 Abcam; isotype control anti-rabbit IgG polyclonal ab171870 Abcam). Protein G Dynabeads were again prepared by washing with RIPA buffer and 100 μl slurry was added per IP, rotating on nutator for 1 h at 4°C. IPs were washed four times with supplemented RIPA buffer. Final beads were eluted in elution buffer (50 mM Tris–HCl pH 7.2, 5 mM EDTA, 1% SDS, 10 mM DTT) and proteinase K (NEB). Reverse crosslinking was performed on the thermomixer shaking at 1200 rpm, first at 60°C for 20 min to allow proteinase K to work, and then at 70°C for an additional 25 min. RNA was isolated using a standard Trizol (Life Technologies) RNA extraction.

The lung cancer cell lines H838, H1975, H1650, H1339, and HCC827 were cultured to approximately 85% confluence on Corning® cell culture flasks with a surface area of 75 cm² and harvested separately. Cells were counted. Cells were washed two times with cold phosphate buffer saline (PBS), making sure to remove any remaining PBS after the second rinse. Then, we added 1 mL cold RIPA Buffer (Product No. 89900, Invitrogen, Darmstadt, Germany) for every 5 × 10⁶ cells and kept on ice for 5 minutes. We added a protease inhibitor cocktail (Product No. 78410, 78420 Invitrogen, Darmstadt, Germany) to the RIPA Buffer immediately before use. Protein was extracted from the monolayer-cultured mammalian cell lines as indicated by the manufacturer.
Protein concentrations were quantified using a total BCA Protein Assay Kit (Product No. 23225, Invitrogen). The cell lysates were saved and used for downstream ELISAs and Western blotting. Cell culture supernatants were also collected at different time points and stored at -80°C.
A human total Erythropoietin Receptor ELISA Kit (DYC963-5) and human VEGF DuoSet ELISA Kit (R&D Systems GmbH. Wiesbaden, Germany) were used to measure the levels of EPO-R in the cell lysates and VEGF in cell culture supernatants, according to the manufacturer's instructions.

Immunoblotting was performed according to Invitrogen protocols for Mini Gel Tank and iBlot2 dry system. In brief, cell lysates were prepared to incubate cells with RIPA buffer (Invitrogen) containing Halt Protease Inhibitor Cocktail (Invitrogen) and quantified using BCA assay (Thermo Fisher Scientific). EVs lysates were prepared incubating cells with 50 μl of RIPA buffer (Invitrogen) containing Halt Protease Inhibitor Cocktail (Invitrogen) and sonicated for 10 min. In all, 25 μl of EVs lysate was loaded on the gel. Proteins were separated using NuPAGE Bis-Tris 4–12% gels (Invitrogen) and transferred to nitrocellulose membranes using the iBlot2 dry method. Membraned were blocked for 40 min in 4% milk and incubated with the primary antibodies in 4% milk 1.5 h at room temperature for anti-CHMP2A antibody or ON at 4 °C for all the other antibodies used. After incubating the membrane with the appropriate secondary antibody conjugated to horseradish peroxidase, protein levels were detected using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore). CHMP2A overexpression lysate (OL) control used in Fig. 2c was purchased from Origene. CHMP2A Antibody, rabbit polyclonal, Proteintech, cat. no. 10477-1-AP (1:600). GAPDH Loading Control Monoclonal Antibody (GA1R) (1:1000), Invitrogen, cat. no. MA5-15738. CD9, clone (D8O1A) Rabbit mAb, Cell Signaling, cat. no. 13174S (1:1000).

ZO-1 and occludin in the colon were assessed by a Western blot. RIPA buffer (Thermal Scientific, USA) was used to homogenize the colon tissue. Then, the sample was centrifuged and prepared to measure the protein concentrations by BCA kits (P1511, Applygen Technologies Inc., Beijing, China). Each sample was loaded to the 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. After being blocked for 4 h in a Tris-buffered saline/Tween 20, the membrane was incubated with primary antibodies of ZO-1 (1:1000; ab96587, Abcam), occludin (1:1000; ab216327, Abcam), and β-actin (1:5000; ab8227, Abcam) at 4 °C overnight. After that, the membrane was incubated with the secondary antibody. The immunoreactive protein bands were visualized using a Clarity Western ECL substrate kit (Sigma, CA, USA). Scion Image (Frederick, MD, USA) was used to perform the densitometry analysis of protein bands. The values were normalized against the intensity of β-actin (Figure 1).