Analysis of the interactome of LAT in CRISPR/CAS9 modified mouse CD+ T cells

Using a CRISPR/Cas9-based approach we engineer primary CD4+ T cells in which the LAT protein was tagged with an affinity Twin-Strep-tag (OST) with the purpose of determining by quantitative mass spectrometry the composition and dynamics of the signalosome assembling around LAT prior to and following T cell activation. Affinity purification of the OST tagged protein was performed using Streptactin beads, from T cells left non-stimulated, or stimulated for 30s or 120s with anti-CD3 and anti-CD4 antibodies. Each AP-MS purification is associated with a corresponding control (purification from non-edited WT CD4+ T cells, cultured and stimulated in the same conditions). The number of replicate biological experiments was n=3 for all conditions (time-points), and each sample was analyzed once by single-run nano LC-MS, resulting in 18 raw files.