Immunoglobulin g igg

METTL3-including complexes were immunoprecipitated from cellular lysate obtained after sonication and the use of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) buffer (40 mM HEPES [pH 7.4], 120 mM NaCl, 1% CHAPS, and 1 mM EDTA, supplemented with protease and phosphatase inhibitors). IPs were performed using the Catch and Release v2.0 reversible IP system (Merck, France) and anti-METTL3 (Abcam, France). Immunoglobulin G (IgG) (Abcam, France) was used as a control. Elutions from IP were performed using the non-denaturing elution buffer according to the manufacturer’s instructions. Then, 30 μL of elution was used in the METTL3 enzymatic assay. The METTL3 enzymatic assay was conducted in reaction buffer (20 mM Tris [pH 7.5], 1 mM DTT, 0.01% Triton X-100, and 40 U/100 mL of RNaseOUT buffer). The reaction mixture contained unmethylated mimic miR-200b-3p with biotin tag and S-adenosyl methionine (SAM). Enzymatic assay reactions were incubated overnight at room temperature on a shaker. After streptavidin isolation, the presence of N6-adenosine methylation was determined by a dot blot. Dots were then incubated with anti-m6A and anti-adenosine (as a loading control) antibodies overnight. For signal detection, secondary horseradish peroxidase (HRP) antibodies were used and signal was detected on ChemiDoc MP (Bio-Rad, France).

Enucleated eyes were fixed in formaldehyde (Super Fix, Kurabo, Osaka, Japan) for 7 days and then embedded in paraffin (Sigma-Aldrich) [20 (link),21 (link)]. paraffin sections were sliced into 10 µm-thick sections and prepared as a series of five sequential slides with an autoslide preparation device (Kurabo). We first performed H&E staining before IHC evaluation. Retinal sections were blocked with 5% goat serum in PBS for 1 h at room temperature. Primary antibodies to the following antigens were applied: CD3 (host: rabbit, Abcam, Cambridge, UK), Iba1 (host: rabbit, Wako, Osaka, Japan), MHC class II (MHC-II; host: mouse, DakoCytomation, Glostrup, Denmark), CD20 (host: rabbit, Abcam), immunoglobulin G (IgG; host: rabbit, Abcam), NKG2A (host: rabbit, Abcam), and IFN-γ (host: mouse, R&D Systems, Minneapolis, MN, USA). All of the antibodies were incubated at 4 °C overnight. After washing, sections were incubated with secondary antibodies for 1 h at RT and counterstained with DAPI (Life Technologies, Carlsbad, CA, USA). Images were acquired using a confocal microscope (LSM700, Zeiss, Jena, Germany) [20 (link),21 (link)].

Chondrocytes were seeded onto 6-well plates at the density of 2.0 × 10⁵ cells/well and were transfected with miR-NC or miR-671-5p. At 48 h after transfection, RIP assay was conducted using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). Chondrocytes were disrupted with RIP lysis buffer for 5 min on the ice. RNA samples were then incubated with antibody-coated magnetic beads. The antibody against Argonaute2 (AGO2) or Immunoglobulin G (IgG) was purchased from Abcam. RT-qPCR was implemented to analyze RNA enrichment.