Immunoprecipitation

The 2D12 T cell hybridomas expressing FLAG-CIN85 or its mutants with and without TCR stimulation (cross-linking with anti-CD3ε, anti-CD28, and GAH Abs) were lysed and treated with anti–FLAG-M2 Ab-conjugated magnetic beads (Sigma-Aldrich, M8823). Anti–His-tag Ab-conjugated magnetic beads (Sigma-Aldrich, H9914) were used as the negative control. For immunoblotting, the precipitated proteins were released from the beads by boiling in SDS sample buffer. For MS analysis, the precipitated proteins were released from the magnetic beads using the elution buffer by rotating for 20 min at room temperature. The Sts-2–GFP– or cCbl-GFP–expressing 2D12 T cell hybridomas were activated and lysed as mentioned above, and the immunoprecipitations were done using anti-GFP Ab-conjugated magnet beads (MBL, D153-11).