Anti mgea5

For immunoblotting, tissue samples were obtained from each mouse, and protein concentrations were measured with the Bradford assay. Proteins were loaded, separated by a 10% SDS-polyacrylamide gel, and then transferred to a PVDF membrane. The membrane was blocked with Tris-buffered saline with 0.1% Tween 20 (TBST) containing either 0.5% skim milk or 0.5% BSA for one hour at room temperature. After blocking, membranes were incubated with primary antibodies, anti-MGEA5 (Cat #: 14711-1-AP, Proteintech, 1:1000), anti-O-GlcNAc (RL2) (Cat #: MA1-072, ThermoFisher, 1:1000), anti-HA-Tag (Cat #: 3724, Cell Signaling, 1:1000), or anti-β-actin (Cat #: Novus Biologicals, Novus, 1:50000) overnight at 4 °C. After washing six times with TBST, the membrane was incubated with secondary antibodies, anti-rabbit IgG-HRP (Cat #: SC-2004, Santa Cruz, 1:5000) or anti-mouse IgG-HRP (Cat #: 62-6520, ThermoFisher, 1:5000) for one hour at room temperature. Signals were visualized using an enhanced chemiluminescence (ECL) detection kit (Millipore, Burlington, MA, USA).

Paraffin-embedded 5-μm TMA sections were deparaffinized in xylene and rehydrated via soaking in decreasing percentages of ethanol solutions (100% twice, 90% and 70% once). Sections were heated in 0.1 M citrate for antigen retrieval, treated with 3% H₂O₂ to block endogenous peroxidase activity, and incubated overnight at 4°C with anti-OGT (1:50; ProteinTech, Chicago, IL), anti-MGEA5 (1:100; ProteinTech), or anti-O-GlcNAc (1:200; Thermo Fisher Scientific, Waltham, USA) primary antibodies. After washing in phosphate-buffer saline (PBS), sections were incubated with peroxidase-labeled secondary antibody for 1 h at room temperature. Sections were then incubated with diaminobenzidine, washed, and counterstained with hematoxylin. All stains were examined by pathologists and semi-quantitatively scored as follows: 0 (no staining), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive); percentage scores were 0 (0%), 1 (≤10%), 2 (11–50%), and 3 (51–100%). The IHC score for each specimen represents the intensity score multiplied by the percentage score, which ranged from 0–9.