Fig. 4.

Schematic representation of a 3-plex SILAC approach for profiling phosphorylation dynamics of SLP-65 in resting and activated B cells. DT40 B cell transductants expressing SLP-65 with a N-terminal One-STrEP-Tag (♦) were cultured in medium with light, intermediate, or heavy amino acids as indicated and left untreated or BCR-activated for 2 or 20 min. Lysates were mixed in a 1:1:1 ratio. SLP-65 was affinity-purified, subjected to 1D-PAGE, and digested with trypsin in the isolated gel slice. Resulting phosphopeptide products were enriched by TiO₂ chromatography and analyzed by LC-MS/MS. See “Results” for details.