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).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.