Odyssey v1

For cellular protein lysates, cells were scraped on ice using cold Ripa lysis buffer (150 nM NaCl, 50 mM Tris–HCl pH 8, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail (CompleteTM, Roche), 1 mM Na3VO4 (Sigma), 100 mM NaF (Sigma), and 1 mM DTT (Sigma).
Proteins were separated in 4–20% SDS–PAGE (Criterion Precast Gel, Bio‐Rad) and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% dried milk in TBS‐0,1% Tween 20 or in Odyssey Blocking Buffer (LI‐COR, Biosciences) and incubated at 4°C overnight with primary antibodies. The list of primary antibodies is provided in Appendix Table S5.
Membranes were washed in TBS‐0,1% Tween 20 and incubated 1 h at RT with IR‐conjugated (AlexaFluor680, Invitrogen or IRDye 800, Rockland) secondary antibodies for infrared detection (Odyssey Infrared Detection System, LI‐COR) or with the appropriate horseradish peroxidase‐conjugated secondary antibodies (GE Healthcare) for ECL detection (Clarity Western ECL Substrate, Bio‐Rad). Band quantification was performed using the Odyssey v1.2 software (LI‐COR) or the QuantiONE software (Bio‐Rad Laboratories). The Re‐Blot Plus Strong Solution (Millipore) was used to strip the membranes, when reblotting was needed.

Immunoblots were performed on extracts from primary neuronal cultures or from cortex collected in lysis buffer (20 mM HEPES, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 2.5 mM EGTA, 0.1 mM dithiothreitol, 50 mM NaF, 1 mM Na₃VO₄, 1% Triton X-100 and a protease inhibitor cocktail (Sigma;11873580001)^{29 (link)}. Protein concentration was determined using a Bradford assay. Proteins (20–40 µg) were separated on 10, 12 or 15% polyacrylamide gels and analyzed by immunoblotting. Antibodies were diluted in the blocking solution containing 0.1% casein (Sigma; C8654). Primary antibodies used were: anti-ATG7 (sc-33211) rabbit polyclonal and anti-BECN1 (sc-11427) rabbit polyclonal from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-LC3 (ab48394) rabbit polyclonal from Abcam (MA, USA); anti-cleaved CASP3/caspase-3 (9661) rabbit polyclonal from Cell Signalling Technology (MA; USA); anti-SQSTM1 (P0067) rabbit polyclonal from Sigma; anti-FODRIN/SPECTRIN (FG6090) mouse monoclonal from Enzo Life Sciences and anti-ACTA/α- ACTIN (MAB1501) mouse monoclonal from Millipore/Merck (MA, USA). Secondary antibodies were polyclonal goat anti-mouse or anti-rabbit IgG from LiCOR (IRDye 680 or IRDye 800). Protein bands were visualized with the Odyssey Infrared Imaging System (LICOR, NE, USA). Odyssey v1.2 software (LICOR) was used for analysis. Values were normalized with respect to ACTIN.

The proteins were extracted from human ccRCC cells and specimens. Lysate was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gel was blotted onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was blocked in 5% nonfatmilk, and then incubated with either rabbit anti-human PEDF (ab180711, Abcam, USA), Bcl-2 (sc-56015, Santa, USA), Bax (sc-20067, Santa, USA), MMP9 (sc-53630, Santa, USA), MMP2 (sc-13595, Santa, USA), or Actin (3700, Cell Signaling Technology, USA). After washing, the membrane was incubated with the fluorescence-conjugated anti-mouse or anti-rabbit IgG (Invitrogen, USA). The bound secondary antibody was quantified using the Odyssey v1.2 software (LI-COR, USA) by measuring the band intensity (area×optical density) for each group and then normalized with Actin. The final results are expressed as fold changes by normalizing the data to control values.

The total amount of protein was extracted from HUVECs, 4T1 breast cancer cells and tumor tissues in each group for immunoblotting analysis. Briefly, the protein concentrations were determined with a bicinchoninic acid protein assay kit using bovine serum albumin as the standard. Equal amounts of protein (100 μg) were fractionated by SDS-PAGE and blotted to PVDF membrane (Millipore, Bedford, MA). The PVDF membrane was blocked with 5% non-fat milk and incubated with the primary antibody at 4 °C overnight. Then the blots were incubated with secondary antibody: Alexa Fluor® 800 goat anti-mouse or anti-rabbit IgG (Invitrogen) for 1 h at room temperature. The primary antibodies against VEGFR2, Akt, phosphorylated of Akt (p-Akt), Bcl-2, Bax, MEK, p-MEK, ERK, p-ERK, Raf and GAPDH were purchased from Abcam and Cell Signaling. Western blot bands were captured by using the Odyssey Infrared Imaging System (LI-COR Biosciences) and quantified with Odyssey v1.2 software (LI-COR Biosciences). GAPDH was used as the internal control. Western blotting experiments were repeated four times.

Exosomes or cells were lysed with RIPA buffer containing a complete protease inhibitor tablet (Roche, Switzerland). Lysate was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gel was blotted onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was blocked in 5% nonfatmilk, and then incubated with either rabbit anti-human E-cadherin (3195, Cell Signaling Technologies, USA), N-cadherin (14,215, Cell Signaling Technologies, USA), Vimentin (5741, Cell Signaling Technologies), Twist (ab49254, Abcam, USA), PTEN (ab32199, Abcam, USA), CD103 (GTX64393, Genetex, USA), CD9 (20597–1-AP; Proteintech, USA), or Actin (3700, Cell Signaling Technology, USA). After washing, the membrane was incubated with the fluorescence-conjugated anti-mouse or anti-rabbit IgG (Invitrogen, USA). The bound secondary antibody was quantified using the Odyssey v1.2 software (LI-COR, USA) by measuring the band intensity (area × optical density) for each group and then normalized with Actin. The final results are expressed as fold changes by normalizing the data to control values.