Transblot device

VLP samples and cell lysates were separated by SDS-PAGE (15 μg of protein per lane for the cell lysates, and five times the cell lysate volume for the VLP samples) and transferred to a membrane using a transblot device (BioRad, Hercules, CA, USA). VP40 was detected using primary antibody F56-6A1.2.3 (Thermo Fisher Scientific) and secondary antibody AB 6808 conjugated to horseradish peroxidase (Abcam, Cambridge, UK). GAPDH was detected using primary antibody AB 8245 (Abcam) and secondary antibody AB 6808 as above. Signals were detected using the enhanced chemiluminescence substrate according to the manufacturer’s instructions (Thermo Fisher Scientific). The percentage of budding was determined by FIJI analysis [46 (link)].

To test the quality and specificity of the sera produced, we loaded 2 and 10 μg of the recombinant protein and total protein from cell culture samples, respectively, mixed with 2 × Laemmli sample buffer, on two 12.5% SDS-PAGE gels, one was stained them with Coomassie blue after electrophoresis, the other one was used to transferprotein bands onto a PVDF membrane (Mini ProBlott Membranes, Applied Biosystems, Foster City, CA, USA) using a Transblot device (Bio-Rad). Membrane strips was subsequently blocked for 30 min at room temperature in a TBS solution containing 1% casein and 0.05% Tween 20. After blocking, the membranes were incubated with chicken anti-rCSUI_001473 polyclonal sera, or negative chicken sera dilutions 1:500 in TTBS buffer (100 mM Tris, 0.9% NaCl, 0.1% Tween 20) at room temperature for 1 h. After rinsing with TTBS for 30 min, blots were exposed to biotinylated goat anti-chicken IgY (Vector Laboratories, Burlingame, CA, USA) as secondary antibody at 1:5000 dilution in TTBS buffer for 1 h at room temperature, incubated with avidin–biotin complex solution (Vector Laboratories) and finally detected by addition of 3,3′-5,5′-tetramethylbenzidine according to the manufacturer’s instructions (Vector Laboratories).

Western blot analyses of both cell lysates and EVs were carried out after resolving samples in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In brief, the analysis on cell lysates was performed by washing cells twice with 1× PBS (pH 7.4) and lysing them with 1× SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE and transferred by electroblotting on a 0.45 μM pore size nitrocellulose membrane (GE Healthcare Europe GmbH, Milan, Italy) overnight using a Bio-Rad (Hercules, CA, USA) trans-blot device. For Western blot analysis of EVs, they were lysed and analyzed as described for cell lysates. For immunoassays, membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Triton X-100 for 1 h at room temperature, then incubated overnight at 4 °C with specific antibodies diluted in PBS containing 0.1% Triton X-100. Filters were revealed using 1:1000-diluted sheep anti-Nef antiserum ARP 444 (MHRC, London, UK), 1:500-diluted anti-β-actin AC-74 mAb from Sigma (St. Louis, MI, USA), 1:500 diluted anti-Alix H-270 polyclonal Abs from Santa Cruz (Dallas, TX, USA), and 1:1000 diluted 6AT18 anti-6×His-tag mAb (Sigma).

Briefly, 20 μg of total protein from tissues were extracted as described elsewhere⁵⁰ , separated by electrophoresis in SDS–polyacrylamide gels and transferred to a nitrocellulose membrane in a semi-dry Trans-Blot device (Bio-Rad, Madrid, Spain). The following primary antibodies were applied: anti-mouse aggrecan (AGG) antibody (1/100; ab3778; Abcam) and anti-mouse type II collagen (COL-II) antibody (1/1,000; ab34712; Abcam) in 3% BSA, overnight at 4 °C. The binding signal was detected by chemiluminescence using Horseradish Peroxidase-linked secondary antibodies. EZBlue gel staining reagent (Sigma-Aldrich) was used for tissue protein loading control. Densitometric measurements were normalized relative to total protein presence in femoral condyles with AC using Quantity One software (Bio-Rad) and expressed as arbitrary densitometric units (A.U.)^{49 (link),50 ,52 (link)}.

Immunoblots were performed using saponin-lysed, infected erythrocytes. Parasite proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) as described previously (63 (link), 64 (link)) and transferred to a nitrocellulose membrane (Amersham Protran; 0.45-μm-pore-size nitrocellulose membrane; GE Healthcare) using a Trans-Blot device (Bio-Rad) according to the manufacturer’s instructions. The membranes were blocked with 3% skim milk in Tris-buffered saline (TBS) for 30 min and then probed with mouse anti-GFP (clone 7.1 and 13.1; 1:1,000, Roche) or rabbit anti-aldolase (65 (link)) (1:2,000). The chemiluminescent signal of the horseradish peroxidase-coupled secondary antibodies (Dianova) was visualized using a Chemi Doc XRS imaging system (Bio-Rad) and processed with Image Lab 5.2 software (Bio-Rad).
To perform loading controls and ensure equal loading of parasite material, rabbit antialdolase (65 (link)) antibodies were used. The corresponding immunoblots were incubated twice in stripping buffer (0.2 M glycine, 50 mM dithiothreitol, 0.05% Tween 20) at 55°C for 1 h and washed three times with Tris-buffered saline for 10 min before reprobing.

Total cell lysates (45 μl, at 30 mg/ml) from yeast having the chimeras expressed from pYeF2 (see above) were mixed with 15 μl of loading buffer (TAE 2X, 20% glycerol, 8% sarkosyl, 0.5 g/l bromophenol blue, plus protease inhibitors). Samples were incubated at RT for 10 min, and electrophoresis performed in 1.5% agarose gels (TAE 1X, 0.1% SDS) at 100 V for 7.3 h, 10∘C (Molina-García and Gasset-Rosa, 2014 ). Proteins were then transferred to a PVDF membrane in a Trans-Blot device (Bio-Rad) in TAE 1X, 0.1% SDS, at 16 V for 15 h, 10∘C. Detection was performed with anti-HA (1:1,000).