Extended Data Table 2 |.
Targeted mass list for IgE glycosylation sites N140, N168 and N265 glycopeptides from the chymotryptic digest and N218, N371 and N394 glycopeptides from the tryptic digest
| FACS assay | Target | Clone | Fluorochromes | Vendor | Dilution |
|---|---|---|---|---|---|
| IgE loading on mouse skin mast cells | Mouse CD45 | 30-F11 | APC/Cyanine7 | Biolegend | 1:400 |
| Mouse/human CD11b | M1/70 | PerCP/Cyanine5.5 | Biolegend | 1:100 | |
| Mouse CD11c | N418 | FITC | Biolegend | 1:100 | |
| Mouse Ly-6G/Ly-6C (Gr-1) | RB6–8C5 | FITC | Biolegend | 1:100 | |
| Mouse CD117 (c-Kit) | 2B8 | APC | Biolegend | 1:100 | |
| Mouse IgE | RME-1 | PE | Biolegend | 1:100 | |
| IgE loading on human LAD2 cells | Human CD117 (c-kit) | 104D2 | PE | Biolegend | 1:400 |
| Ovalbumin | A647 | Invitrogen | 1:400 of 2 mg/mL stock | ||
| Human IgE | MHE-18 | APC | Biolegend | 1:400 | |
| Basophil activation tests | Human HLA-DR | L243 | PE/Cy7 | Biolegend | 1:35 |
| Human CD123 | 6H6 | PE | Biolegend | 1:30 | |
| Human CD63 | H5C6 | FITC | Biolegend | 1:30 | |
| Phenotype staining for human mast cells | Human CD117 (c-kit) | 104D2 | APC | Biolegend | 1:400 |
| Human CD45 | HI30 | FITC | Biolegend | 1:100 | |
| Human FcεRIα | AER-37 (CRA-1) | PE | Biolegend | 1:400 | |
| General | Viability | eFluor450 | eBioscience | 1:500 | |
| ELISA assay | Target | Catalog # | Conjugation | Vendor | Dilution |
| Mouse IgE ELISA | mlgE | A90–115A | No | Bethyl | 1:200 |
| mlgE | A90–115P | HRP | Bethyl | 1:30,000 | |
| Human IgE ELISA | hlgE | A80–108A | No | Bethyl | 1:200 |
| hlgE | A80–108P | HRP | Bethyl | 1:30,000 | |
| Immunoblotting | Target | Catalog # | Species/conjugation | Vendor | Dilution |
| Immunoblotting for Syk | Phospho-Syk | 2701 | Rabbit | Cell Signaling Technology | 1:2000 |
| Total | 2712 | Rabbit | Cell Signaling Technology | 1:2000 | |
| Secondary antibody | Rabbit IgG | W4011 | HRP | Promega | 1:30,000 |
| Immunoblotting for Actin | Actin | SC-47778 HRP | HRP | Santa Cruz Biotechnology | 1:50,000 |
| Passive Anaphylaxis | Species | Clone | Antigen specificity | Vendor | |
| IgE | Mouse | SPE-7 | dinitrophenyl (DNP) | Sigma-Aldrich | |
| IgE | Mouse | MEA-36 | Trinitrophenyl (TNP) | Biolegend |