R0278

Proteins were prepared with a detergent buffer (R0278, Sigma-Aldrich), and the protein concentration was determined using the Pierce™ BCA Protein Assay Kit (20164, Thermo Fisher Scientific)y. Equal amounts (60 μg) of protein samples were separated by a 12% gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 1610174, Bio-Rad Laboratories, Shanghai, China) and transferred onto polyvinylidene fluoride (PVDF; IPVH00010, Millipore, Bedford, MA, USA) membranes. After block by skim milk (37587, Thermo Fisher Scientific) for 1 h, the samples were then incubated with respective primary antibodies including anti-RNF157 (WH0114804M1), anti-PLRG1 (SAB2500805), anti-SMU1 (SAB1407636), anti-CHD1 (ZRB1692) and anti-PSMD8 (SAB1406325) provided by Sigma-Aldrich, and anti-β-actin (PA1-46296), anti-CD63 (MA1-19281), anti-TSG101 (1062BD), anti-calnexin (PA5-34665), anti-HDAC1 (49-1025), anti-RAN (48-2300), anti-EMD (701503) and anti-FXR2 (MA1-5773) provided by Invitrogen overnight at 4°C, followed by cultivation with secondary antibodies marked by horseradish peroxidase (HRP) (32260, Invitrogen) at room temperature for 2 h. Eventually, enhanced chemiluminescence (ECL) detection system (32209, Thermo Fisher Scientific) was applied for gray-scale value analysis.

To preserve the secondary structure of APN, serum (1 µl) from FGF21 (PF-05231023) vs. vehicle-treated Phase I retinopathy mice was incubated with Laemmli’s SDS sample buffer (BP-110R; Boston BioProducts Inc.) for 1 h at room temperature [40 (link)]. Primary antibody against APN (1:1000, AF1119; R&D) was used. Signals were visualized with 1:5000 corresponding horse-radish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Pierce). The band signal intensity was quantified using Image J. Seven mice per group were used.
Two retinas from the same mouse were pooled for protein extraction using RIPA lysis buffer (R0278, Sigma-Aldrich) with protease inhibitor (1:1000, P-8340, Sigma-Aldrich) and phosphatase inhibitor (1:100, P0044, Sigma-Aldrich). Primary antibodies targeting phospho-TFEB (Ser142) (1:1000, ABE 1971, EMD Millipore), TFEB (1:2000, A303-673A, Bethyl Laboratories) were used. Signals were visualized with 1:5000 corresponding horse-radish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Pierce). β-ACTIN (1:2500, A1978, Sigma-Aldrich) was used as internal control. The signal of band intensity was quantified using Image J. The levels of target proteins were first referred to the levels of β-ACTIN. Ratio of change was then calculated referring to the vehicle control group as one. Six mice per group were used.

The following reagents were obtained: ketamine (Dopalen injectable^®, Paulínia, Brazil), xylazine (Anasedan injectable^®, Paulínia, Brazil), Tween 80 (Synth^®, Diadema, SP, Brazil), hexadecyltrimethylammonium bromide (HTAB) (Sigma Aldrich^® H5882, Steinheim, Germany), o-dianisidine dihydrochloride (Sigma Aldrich^® D3252, Steinheim, Germany), dextran sulphate sodium salt (DSS)—colitis grade (36,000–50,000 MW, cod. 02160110-CF, MP Biomedicals, LLC, Solon, OH, USA), hydrogen peroxide (Synth^®), horseradish peroxidase (Sigma Aldrich^® P8250, Steinheim, Germany), trichloroacetic acid (TCA) (Sigma Aldrich^® T4885, Steinheim, Germany), reduced L-glutathione (Sigma Aldrich^® G-4251, Steinheim, Germany), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) (Sigma Aldrich^® N-7505, Steinheim, Germany), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma Aldrich^® D-8130, Steinheim, Germany), and buffer RIPA (Sigma Aldrich^® R0278, Steinheim, Germany). IL-1β mouse, IL-10 mouse, CXCL-MIP-2 mouse, and TNF-α mouse were obtained from R&D Systems^®, Inc. (Minneapolis, MN, USA).

Tissue samples were homogenized in radio‐immunoprecipitation assay (RIPA, R0278, Sigma) buffer mixed with 1% protease inhibitor cocktail (P8340, Sigma). Protein concentration was measured by Bradford assay. Equal amounts of protein were loaded for gel electrophoresis. After blotting, membranes (Hybond‐P, GE Healthcare) were probed with rabbit polyclonal anti‐ESAM antibody (1:1000, ab74777, Abcam), followed by incubation with HRP linked secondary antibodies (1:5000, 7074 S, Cell Signaling). Enhanced chemiluminescence was visualized autoradiographically by ChemiDoc XRS+ (Bio Rad). Protein expression was normalized for the total loaded protein using a BIO‐RAD TGX Stain‐Free FastCast Acrylamide Kit (Cat# 1610183), ChemiDoc imaging system, and Image Lab Software (Bio Rad).

After the PET scan, the animals were transcardially perfused with cold phosphate-buffered saline pH 7.4 (PBS), and the brain was removed for tissue analysis. The frontal cortex and hippocampus were excised from the brain, placed in an ice-cold PBS solution, snap-frozen in liquid nitrogen, and stored at − 80 °C until further analysis. RIPA buffer (Sigma-Aldrich, R0278 – containing 150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) was added to the brain tissue (50-μl/mg tissue) and cooled on ice. The tissue was pounded until no solid fragments were visible anymore. The homogenized tissue was centrifuged at 12,000 rpm for 15 min. The supernatant was collected for total protein quantification by the bicinchoninic acid assay (BCA) using bovine serum albumin as a standard. Then, BDNF was measured with ELISA (Cloud-clone, SEA011Ra) according to the manufacturer instructions. Intra-assay precision was < 10%. Tissue lysate was diluted 1:5 in PBS (five samples were diluted 1:6 due to low amount of lysate). Samples were read at 450 nm and corrected for the total amount of protein.