Bovine serum albumin (bsa)

Cells were lysed in an LDS sample buffer (NuPAGE, Novex, Life Technologies, Carlsbad, CA, USA), sonicated, and boiled at 96 °C for 3 min. Proteins were separated on 4–20% gradient SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore, Burlington, MA, USA). Membranes were blocked in TBS-T (Tris Buffered Saline with 0.1% Tween 20) containing 5% Bovine Serum Albumin (Euromedex, Souffelweyersheim, France) or 5% skim milk and incubated with primary antibodies overnight at 4 °C. Membranes were washed and then incubated at room temperature with Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, Ely, UK). After washing, detection was performed by chemiluminescence (Clarity^TM Western ECL substrate, Bio-Rad). Image acquisition and quantification of immunoblots were performed with a Fusion Solo X chemiluminescence imaging system using the Evolution Capture software (Vilber Lourmat, Marne La Vallée, France).

After bear serum incubation, cells were scraped into 200 μl of ice-cold lysis buffer (TRIS-HCl 20 mM, NaCl 138 mM, KCl 2.7 mM, MgCl₂ 1 mM, Glycerol 5%, NP 40 1%, EDTA 5 mM, Na₃VO₄ 1 mM, NaF 20 mM and DTT 1 mM) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, France). Protein concentration was determined by Bradford quantification. Western blotting was performed, as described previously^{29 (link)}, loading 20 μg of total protein on precast gels (Mini-protean TGX Stain-free^TM gel, Biorad, France). After migration, gels were UV exposed for 3 minutes and pictures were taken for further quantification of protein loading. After semi-dry transfer, all membranes were blocked with 4% BSA (Bovine Serum Albumin, Euromedex, Souffelweyersheim, France) before incubation with primary antibodies (see Table S1). Corresponding secondary HRP antibodies were used for chemiluminescence revelation (Chemidoc Bio-Rad).

Horse heart Myoglobin (Met-Myoglobin, metMb) was purchased from Sigma-Aldrich (purity ≥ 90%). It was purified before use, following the procedure described below. Deuterated water (Sigma-Aldrich, 99.9% isotopic purity) and glutaraldehyde (TCI, 50% in water w/w) were kept under nitrogen atmosphere to avoid contamination by water vapor after first opening. Polypropylene sheets (4.75 µm  ±  3%, Spex Sample Prep) and steel foil (5 µm) were used for protein hydrogel preparation and irradiation. Bovine serum albumin (Euromedex, > 98%), Human serum albumin (Sigma-Aldrich), and β-lactoglobulin from bovine milk (Sigma-Aldrich, ≥ 90%, mixture of 2 variants) were used after similar purification to that described for myoglobin.

Immunoblotting was performed following standard procedures. Briefly, 10 μg of protein were separated on NuPAGE Novex Bis-Tris 4–12% pre-cast gels (Invitrogen-Life Technologies, Carlsbad, CA, USA) and transferred to Immobilon polyvinylidene difluoride membranes (Merck-Millipore, Darmstadt, Germany). Unspecific binding was reduced by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% w/v bovine serum albumin (Euromedex, Souffelweyersheim, France). Following, membranes were probed with antibodies specific for CFTR (Thermofisher) and beta-Actin (Abcam). Primary antibodies were revealed with species-specific immunoglobulin G conjugated to horseradish peroxidase (Southern Biotech, Birmingham, AL, USA), followed by chemiluminescence analysis with the SuperSignal West Pico reagent by means of an ImageQuant 4000 (GE Healthcare, Little Chalfont, UK).

Cells were plated in 24-well plates and treated for 24 h with Hyabak, Thealose Duo or trehalose and CQ was either added or not added, 4 h before lysis to study the autophagic flux. Cells were washed with PBS and lysed in 65 mM Tris, pH 6.8, 4% sodium dodecyl sulfate (SDS), 1.5% β-mercaptoethanol, and placed at 100°C for 5 min. Protein lysates were resolved on 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), then electrotransferred to a polyvinylidene fluoride membrane (Amersham Plc., Buckinghamshire, United Kingdom). Membranes were blocked for 1 h in PBS 0.1% Tween containing 5% bovine serum albumin (04-100-812-C; Euromedex, Strasbourg, France) before antibody staining overnight at 4°C. HRP-labeled secondary antibodies were used followed by chemiluminescence detection according to the manufacturer's instructions (Immobilon; Millipore). Protein levels were quantified using ImageJ software.

Endothelial cells from coronary arteries were isolated from porcine left circumflex coronary arteries by collagenase treatment (type I, Worthington, 1 mg/ml for 12 min at 37°C), and cultured in culture dishes containing medium MCDB 131 (Invitrogen, Saint Aubin, France) supplemented with 15% fetal calf serum, penicillin (100 U/ml), streptomycin (100 U/ml), fungizone (250 µg/ml), and L-glutamine (2 mM) (all from Cambrex, Wiesbaden, Germany), and grown for 48–72 h. Cells (first passage) were exposed to serum-free culture medium in the presence of 0.1% bovine serum albumin (Euromedex, Souffelweyersheim, France) for 6 h prior to treatment.

Four µm paraffin-embedded kidney sections were stained with Masson’s trichrome for morphological analysis. For immunohistochemistry, 4 µm sections of frozen kidney were fixed in acetone, incubated for 1 h in 5% bovine serum albumin (Euromedex, Souffelweyersheim, France), followed by 1 h 30 min at room temperature with biotinylated mouse anti-human IgA (Catalogue number 555884) or anti-mouse CD11b (Catalogue number 553309) (both from BD Biosciences, Le Pont de Claix, France). Detection was performed with vectastain elite ABCkit (Vector, Burlingame, CA, USA). Slides were mounted with Immuno-mount (Thermo Fisher Scientific), read with an upright microscope, DM2000 (Leica, Wetzlar, Germany) at ×400 magnification using IM50 software (Leica) and scanned with AperioScanScope CS System (Leica Microsystems SAS, Nanterre France) and staining was quantified using TRIBVN CaloPix software (TRIBVN, Chatillon France). For CD89 kidney staining, 4 µm sections of frozen kidney were fixed in acetone, incubated for 1 h in 5% bovine serum albumin, followed by anti-CD89 mAb (clone A3, homemade) and developed by staining with anti-mouse Ig coupled with FITC. Slides were mounted with Immuno-mount, read with an upright fluorescence microscope (Leica, Wetzlar, Germany) at ×40 magnification using IM50 software (Leica).