Flotillin 1

Antibodies against p-ERK, ERK, Flotillin-1, CD63, β-actin (Santa Cruz Biotechnology, CA, USA), TSGA10 (Sigma-Aldrich, America), CD9, ALIX, TSG101, and actinin-4 (Abcam, MA, USA) were used in western blot as previously described [51 (link)].

An amount of 100 μL of plasma and 3 mL of urine samples were used for EV isolation using ExoGAG. The pellet containing EVs was resuspended in 200 μL of PBS and incubated for 1 h at 4 °C with antibodies against EVs’ protein markers [36 (link)]: CD9 (1:50, sc13118, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD63 (1:50, sc5275, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and flotillin-1 (1:50, sc74566, Santa Cruz Biotechnology, Santa Cruz, CA, USA). An isotype IgG antibody (1:50, 400120, BioLegend, San Diego, CA, USA) was used as negative control and added in equivalent volumes. Subsequently, an anti-mouse Alexa 488 (1:1000, ab150113, Abcam, Cambridge, UK) secondary antibody was added and stained for 1h at 4 °C in the dark. EVs were then washed once with PBS by centrifugation at 3000× g for 15 min at 4 °C and analyzed by flow cytometry (BD FACSAriaTM IIu, BD biosciences, San José, CA, USA).

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.

Western blots were used to analyze the protein composition of P100 pellets. We used primary antibodies: (dilution used for all primary antibodies 1/300) for, GPR177 (MyBioSource, Catalog Number: MBS769833; Abcam, GM130 Catalog Number: ab52649; Thermo Fisher Scientific Inc., Wnt3a Catalog Number: PA5-44946; Abcam, Wnt5a Catalog Number: ab174963; Thermo Fisher Scientific Inc., Wnt7a Catalog Number: PA5-80231. All the following antibodies were from Santa Cruz Biotechnology, Inc., Wnt5a, Catalog Number: sc-365370; Alix, Catalog Number: sc-53540; TSG101, Catalog Number: sc-7694; NCAM-L1 Catalog Number: sc-374046; Flotillin-1, Catalog Number: sc-133153; Tubulin, Catalog Number: sc-8035; CD63, Catalog Number: sc-5275. Lysate of HT-22 cells were homogenized in RIPA buffer (10 mM Tris–HCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with 1 mM PMSF, 7 μg/mL Pepstatin, 5–10 μg/mL Leupeptin, and 10 μg/mL Aprotinin. The protein content of the cell lysate and the P100 were determined by Pierce^TM BCA Protein Assay Kit. An aliquot containing 30 μg of protein in both the lysate and the P100 was mixed with standard sample loading buffer loaded and ran in a 10% acrylamide gel. Proteins were transferred on to a PVDF membrane overnight at 4°C.

Antibodies were purchased from the following suppliers: against Catalase (#14097), phospho-DRP1 (#3455), GAPDH (#2118), CORO1A (#92904), and LC3B (#2775) from Cell Signaling Technology, Danvers MA, USA; against CD81 (#sc-23962), CD63 (#sc-5275), and Flotillin-1 (#sc-25506) from Santa Cruz Biotechnology, Dallas, TX, USA; against β-actin (#A5441) from Sigma-Aldrich, St. Louis, MO, USA; against GFAP (#610565) from BD Transduction Laboratory, San Jose, CA, USA; against von Willebrand factor (VWF) (#ab6994), PECAM-1 (#ab28364) from Abcam, Cambridge, MA, USA; anti OMG (#12701-1-AP) and GABRA1 (#12410-1-AP) from Proteintech Group, Inc., Rosemont, IL, USA; and mitochondrial complex III subunit core 1 from Invitrogen Corporation, Fredrick, MD, USA.

Tear samples or small EVs were denatured with Laemmli buffer 5x without reducing agents (125 mM Tris-HCl (pH 6.8), 5% SDS, 20% glycerol and 0.01% bromophenol blue). Samples were loaded on polyacrylamide gels and proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride Amersham™ Hybond™ membranes (GE Healthcare, Cleveland, Ohio, USA). The membranes were blocked in 5% (m/v) nonfat milk in TBS-T (20 mM Tris, 150 mM NaCl, Tween 0.2%, pH 7.6) and probed with antibody against exosome markers CD63 (1:500; SICGEN, Cantanhede, Portugal) and flotillin-1 (1:250; Santa Cruz Biotechnology, Dallas, Texas) overnight at 4°C. After washing, the membranes were incubated with secondary anti-goat IgG-HRP-linked antibody (1:10,000; Bio-Rad, Hercules, California, USA) or anti-mouse IgG-HRP-linked antibody (1:10,000; Bio-Rad). The immunoreactive bands were detected by enhanced chemiluminescence (ECL) substrate using an imaging system (LAS500, GE Health Life Sciences, Chicago, Illinois, USA).

Dulbecco's modified Eagle's medium (DMEM), phenylmethanesulfonyl fluoride (PMSF), bovine serum albumin (BSA), and peroxidase-conjugated anti-rabbit IgG were obtained from Sigma (St. Louis, MO). The pre-stained protein ladder (64, 49, 37, 26, and 20 kDa) and fetal calf serum (FCS) was obtained from Invitrogen (Carlsbad, CA). TGF-β was purchased from Austral Biologicals (San Ramon, CA). The polyclonal antibodies against early endosome antigen 1 (EEA1), transferrin receptor (TfR), Smad2, fibronectin, lamin B, EGF receptor, flotillin-1, flotillin-2, CD-55, caveolin-1, TβR-I, and TβR-II were obtained from Santa Cruz (Santa Cruz, CA). The rabbit polyclonal antibody to phospho-Smad2 was purchase from Cell Signaling (Boston, MA).