Image lab software version 6

Equal volumes of resuspended plasma EVs sample pellets were loaded onto 4–15% Criterion^™ TGX Stain-Free^™ Precast gels (Cat# 5678083, Bio-Rad) and transferred onto nitrocellulose membranes (Cat# 1704159, Bio-Rad). Membranes went through blocking with 5% nonfat milk in PBS plus 0.1% Tween 20 (PBS-T) for 1 hour at room temperature (RT), incubation with primary antibodies overnight at 4°C, washing three times with PBS-T, incubation in species-specific, horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at RT, washing three times with PBS-T prior to the addition of chemiluminescence substrate (Cat# 37071, Thermo Fisher Scientific). The Western Blot image was visualized in the ChemiDoc^™ MP Imaging System (Bio-Rad), and protein bands were analyzed by Image Lab^™ software Version 6.0.1 (Bio-Rad). The following antibodies were used: anti-CD63 (Cat# PA5–92370, 1:1000; Thermofisher Scientific) and HRP-conjugated goat anti-Rabbit (Cat# 111-035-003, 1:3000; Jackson ImmunoResearch Laboratories).

Cells were lysed with RIPA buffer (Thermo Fischer Scientific) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich) and 1% phosphatase inhibitor cocktail (Sigma-Aldrich), and lysates were disrupted with sonication. Protein concentrations were determined with a DC protein assay (Bio-Rad, Hercules, CA), and equal amounts of protein (30 µg/ml) per lane were subjected to SDS gel electrophoresis. Protein transfer to a nitrocellulose membrane was performed with the semidry Transblot Turbo system (Bio-Rad). Membranes were blocked in Tris-buffered saline (TBS) + 0.5% Tween20 (Sigma-Aldrich) that contained 5% milk powder. Primary and secondary antibodies were diluted in TBS-Tween20 that contained 5% milk powder. Membranes were cut prior to incubation with primary and secondary antibodies. Primary antibodies were incubated overnight at 4 °C, secondary antibodies were incubated for 60 min at RT. All washing steps were performed in TBS-Tween20. Protein bands were visualized with chemiluminescence and ChemiDoc imaging system (Bio-Rad). Image analyses were performed with Image Lab™ Software version 6.0.1 (Bio-Rad). Original images of blots from all the replicates are included in the supplementary information, acquisition was performed when the total length of the ladder was visible. The antibodies used are shown in Table 2.

Extracellular DNA was extracted as previously described by Kaplan et al. with modification [25 (link)]. Biofilms were grown in triplicate in TSB, TSB_glu, TSB_NaCl and TSB_CTX in 24-well polystyrene microtiter plates, in a total volume of 1 mL per well. After 24 h of growth, the liquid was carefully removed, the plates were washed once with 1XPBS (Sigma-Aldrich, St. Louis, MO, USA) and 50 μL of Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. The biofilm-forming cells were scraped off the bottom surface of the wells and were transferred to 1.5 mL microcentrifuge tubes. The tubes were centrifuged at 13,000 rpm for 25 s and 8 μL of each of the supernatants were resolved on 0.8% agarose gels. Densitometric analyses of eDNA were carried out using Image Lab software version 6.0.1(Bio-Rad Laboratories, Hercules, CA, USA).

The preparation of cell extracts for protein analyses, immunoprecipitation and Western blotting procedures have been described [38 (link), 39 (link)]. The preparation of the cell extracts from the paired samples (normal and tumor tissue) from patients with CRC were performed as described below in the xenograft studies. Depending on the molecular weight of the proteins to be analyzed, calnexin, tubulin, or GAPDH were used as loading controls. CA2 was used as a colon normal tissue control [40 (link), 41 (link)]. Densitometric measurements of the bands were performed using the Image Lab™ Software Version 6.0.1 Bio-Rad Laboratories (Hercules, CA, USA), which was provided with a ChemiDoc apparatus. Stain free blot was performed by adding 50 µl of 2,2,2-Trichloroethanol to 10 ml of the SDS-PAGE gel solution. Detection of total protein was made in the ChemiDoc apparatus (Bio-Rad) following the manufacturer's instructions.

One section of the heart was lysed in the complete RIPA lysis buffer and stored at −80 ◦C for immunoblotting analysis. Immunoblotting analyses were performed according to what has been previously published (Reis-Mendes et al. 2021a (link), b (link)). The enhanced chemiluminescence ECL reagents were used to detect immunoreactive bands, according to the manufacturer’s instructions. Digital images were acquired using the ChemiDoc Imaging System version 2.3.0.07 (Bio-Rad, Hercules, CA, USA). The images obtained were analyzed using the Image Lab software version 6.0.1 (Bio-Rad, Hercules, CA, USA). Protein content in the whole cardiac homogenate was quantified using the Bio-Rad DC Protein assay. Protein loading of Western blotting was confirmed by Ponceau S staining, as done by us in other works (Reis-Mendes et al. 2021a (link), b (link)). 

Samples were applied to wells of a 4–20% acrylamide gradient gel. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. To determine the extent of BSA 3MGCylation, the membrane was blocked in 5% milk powder in Tris buffered saline containing 0.05% Tween 20 (TTBS) and incubated with rabbit α-3MGC IgG (1:40,000) for 16 h at 4 °C. The membrane was then washed with TTBS and incubated with goat α-rabbit IgG HRP-conjugated secondary antibody (1:4000 dilution) for 1 h at room temperature. The membrane was then washed and antibody binding was detected via chemiluminescence using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA). Densitometric analysis was performed using tools native to Image Lab software version 6.0.1 (Bio-Rad, Hercules, CA, USA). Relative band intensities were quantified by normalizing against the most intense band on the same blot.

Heart sections were lysed in RIPA buffer through sonication and were kept at −80 °C until analysis. Samples containing 20 µg of protein were then processed, as previously described [27 (link)]. The slot-blot membranes were blocked with 5% (w/v) dry non-fat milk in TBS-T (100 mM Tris, 1.5 mM NaCl, pH 8.0 and 0.5% Tween 20) for 1 h and then incubated for 1 h with primary antibody (rabbit anti-DNP, 1:1000) in 5% (w/v) non-fat dry milk in TBS-T. The membranes were washed three times (10 min each) with TBS-T and incubated for 1 h with a solution of anti-rabbit IgG-peroxidase (1:5000). Immunoreactive bands were detected using the ECL reagents, according to the supplier’s instructions, and digital images were acquired using the ChemiDoc Imaging System version 2.3.0.07 (Bio-Rad, Hercules, CA, USA). The images obtained were analyzed using Image Lab software version 6.0.1 (Bio-Rad, Hercules, CA, USA).