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. 2012 Aug 17;287(41):34091–34100. doi: 10.1074/jbc.M112.371062

FIGURE 2.

FIGURE 2.

SLP-76 is ubiquitinated during TCR signaling. Sequential immunoprecipitation was used to detect SLP-76 ubiquitination in HEK293T cells (A) and anti-CD3 antibody-stimulated Jurkat T cells (B). FLAG-tagged SLP-76 was transfected into HEK293T or Jurkat TAg cells with HA-ubiquitin (Ub). The cells were treated with MG132 for 0–4 h, and Jurkat T cells were then stimulated with anti-CD3 antibody for 15 min. FLAG-SLP-76 was immunoprecipitated (IP) with anti-FLAG antibody (1st IP); two-thirds of the first anti-FLAG immunocomplex was denatured, followed by a second round of immunoprecipitation (2nd IP) with anti-FLAG antibody. The ubiquitination of the first and second anti-FLAG immunocomplexes was detected by Western blotting (WB) with anti-HA and anti-FLAG antibodies. SLP-76 is ubiquitinated in Jurkat T cells (C) and primary T cells (D) upon anti-CD3 stimulation. Cells were pretreated with or without MG132, stimulated by anti-CD3 antibody, and subjected to immunoprecipitation using anti-SLP-76 antibody. SLP-76 ubiquitination was detected by anti-ubiquitin (Lys-48) and anti-SLP-76 antibodies.