Everyblot blocking buffer

For Western blot analysis, retinas were removed 8 weeks after the first injections from each separate group. Samples (n = 16) were processed for Western blot analysis as described earlier [31 (link)]. Protein concentrations were determined using Bradford reagents. Membranes were blocked in EveryBlot Blocking Buffer (BioRad; Hercules, CA, USA) for 5 min at room temperature and were probed at room temperature with anti-HIF1-α (1:2000; Sigma-Aldrich, Budapest, Hungary) and anti-VEGF-A (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. Non-phosphorylated anti-GAPDH (1:20000; Cell Signaling Technology; Danvers, MA, USA) was used as internal control. Membranes were washed in Tris-buffered saline (TBS; pH = 7.5) containing 0.2% Tween. Anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:3000; BioRad; Hercules; CA, USA) was diluted in EveryBlot Blocking Buffer (BioRad; Hercules; CA, USA) and the membranes were incubated for 1 h at room temperature. The antibody–antigen complexes were visualized by means of enhanced chemiluminescence. For quantification of blots, band intensities were quantified by the NIH ImageJ program (National Institutes of Health, Bethesda, MD, USA).

Cells were lysed in RIPA buffer with protease inhibitors (Roche), 1 mM phenylmethanesulfonylfluoride (PMSF), 10 mM NaF and 1 mM Na₃VO₄ by sonication. Extracts were centrifuged at 14,000 r.p.m (18,407 × g) for 15 min. The supernatants were collected and assayed for protein concentration using the Bio-Rad protein assay method. 20 μg of proteins were loaded on 4–12% Criterion^TM XT Bis-Tris Protein Gel (Biorad), transferred by Trans-Blot-Turbo (Biorad), and blocked with EveryBlot Blocking Buffer (Biorad). Primary antibodies were incubated with membranes overnight at 4 °C. Antibody against ORF1p (Sigma, Cat. # MABC1152; 1:10,000) was used and validated as previously published^{60 (link)}, anti-β-actin is commercially available (Sigma, Cat. #A5316; 1:10,000). Membranes were developed with ECL solution (GE Healthcare) and imaged by Image Lab. The relative expression of the ORF1p protein was measured by densitometry using Image J and normalized for the β-actin intensity of the corresponding lane.

Immunoblotting analysis was used to confirm the presence of EV as previously described (Nikravesh et al., 2019). Briefly, following the electrophoretic separation of proteins using precast gels (4%‐15% Mini‐PROTEAN TBX, Biorad, UK), gels were blotted on polyvinylindene disluoride membranes (Fisher Scientific, UK) and blocked with EveryBlot blocking buffer (BioRad, UK). The blots were incubated overnight at 4°C with primary antibodies to Alix (1:1000 dilution, Santa Cruz, USA), Annexin 2 (1:2000 dilution, Abcam, UK), CD9 (1:1000 dilution, Abcam, UK) and calnexin (1:1000 dilution, Abcam, UK). The membranes were incubated with the appropriate secondary antibody, anti‐rabbit for Annexin 2, CD9 and calnexin (1:3000 dilution, Cell Signaling, UK), and anti‐mouse for Alix (1:3000 dilution, Cell Signaling, UK), for 1 h at room temperature. Chemiluminescence detection of bands were images with ChemiDoc XRS+ system (BioRad, UK) by a chemiluminescence reaction using Clarity™ Western ECL substrate (BioRad, UK) and Image Lab software (Life Science Research, BioRad, UK) following supplier's instructions.

To determine how amlexanox affects inflammatory signaling pathways, BV2 microglial cells were pre-treated with amlexanox or vehicle (< 0.1% DMSO) for 1 h, followed by 100 ng/mL LPS for 23 h. Thereafter, total protein was extracted from the BV2 cells using RIPA lysis buffer (iNtRON, Korea). Following centrifugation at 13,000 g for 20 min at 4 °C, the protein concentration in the supernatant was determined by the Pierce BCA protein assay kit (Thermo Fisher Scientific Inc.), according to the manufacturer’s instructions. Equal amounts of protein in each sample were then loaded into sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). After transferring protein from gel to PVDF membrane (Bio-Rad Laboratories), blocking was conducted using everyblot blocking buffer (Bio-Rad Laboratories). The membrane was then incubated overnight with primary antibodies at 4 °C. To detect the antigen–antibody complexes, HRP-conjugated secondary antibodies (Cell Signaling Technology) were diluted 1:1000, and incubated for 1 h at room temperature. All antibodies were diluted in everyblot blocking buffer. The protein bands were visualized using ImageQuant™ LAS 500 (GE Healthcare Life Science). Expression levels of protein were quantified with ImageJ software.

Amlexanox was purchased from Tokyo Chemical Industry (Tokyo, Japan). STAT3 activation inhibitor SPI was purchased from BioVision (Milpitas, CA, USA). LPS from Escherichia coli O111:B4 was obtained from Sigma–Aldrich (St. Louis, MO, USA). The following primary antibodies, which were diluted at 1:1000 ratios in EveryBlot Blocking Buffer (Bio-Rad Laboratories), were used for Western blotting (WB): rabbit anti-COX2 (Cell Signaling Technology), mouse anti-iNOS (Cell Signaling Technology), rabbit anti-p-AKT (Ser473, Cell Signaling Technology), rabbit anti-AKT (Cell Signaling Technology), rabbit anti- IκBα (Cell Signaling Technology), rabbit anti-p- IκBα (Ser32, Cell Signaling Technology), rabbit anti-IKKε (Cell Signaling Technology), rabbit anti-ERK (Cell Signaling Technology), rabbit anti-p-ERK (Thr202/Tyr204, Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti-p-STAT3 (Tyr705, Cell Signaling Technology), rabbit anti-NF-κB p65 (Cell Signaling Technology), rabbit anti-p-NF-κB p65 (Ser536, Cell Signaling Technology), rabbit anti-JNK, rabbit anti-p-JNK (Thr183/Tyr185, Cell Signaling Technology), rabbit anti-p38 (Cell Signaling Technology), and rabbit anti-p-p38 (Thr180/Tyr182, Cell Signaling Technology), and mouse anti-β-actin (Santa Cruz Biotechnology). Other chemicals for Western blotting were obtained from Bio-Rad Laboratories.

Cells were lysed in protein lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich) and PhosSTOP (Roche). The protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo) and SpectraMax microplate reader. After blocking using Everyblot buffer (Bio-Rad), primary antibodies against the following factors were added: Endoglin (Abcam, ab169545), HES1 (CST, 11,988), HES5 (Abcam, ab19411), HEY1 (Gene Tex, GTX118007), DLL4 (CST, 2589), p-AKT (CST, 4060), AKT (CST, 9272), p-ERK (CST, 9101), ERK (CST, 4695), p-P38 (CST, 9211), P38 (CST, 9212), CD31 (Thermo, MA5–29474), OCT-4 (CST, 2750), NANOG (Abcam, ab173368), calponin-1 (Santa, sc-58707), α-SMA (Sigma, A2547), and β-actin (Santa Cruz Biotechnology, sc-47778). Next, HRP-conjugated anti-mouse, anti-rabbit, and anti-goat secondary antibodies diluted 1:3000 in Everyblot blocking buffer (Bio-Rad) were added. The membranes were imaged using Clarity Western ECL Substrate (Bio-Rad).