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

FIGURE 5.

FIGURE 5.

Lys-30 is an SLP-76 ubiquitination site. A, mass spectrometry spectra of a tryptic peptide of SLP-76 containing the diglycine fragment of ubiquitin attached to Lys-30. FLAG-SLP-76 and HA-ubiquitin were transiently transfected into HEK293T cells, followed by MG132 treatment; the transfected FLAG-SLP-76 proteins were isolated from these cells for LC-MS/MS analysis. B, K30R mutation reduces SLP-76 ubiquitination in HEK293T cells. FLAG-SLP-76 (WT or K30R mutant) was transfected into HEK293T cells with HA-ubiquitin (Ub). Ubiquitination of SLP-76 was determined by sequential anti-FLAG immunoprecipitation (IP), followed by Western blotting (WB) with anti-HA antibody. C, K30R mutation reduces SLP-76 ubiquitination in Jurkat T cells upon anti-CD3 stimulation. FLAG-SLP-76 (WT or K30R mutant) was transfected into Jurkat TAg cells with HA-ubiquitin. The transfected cells were treated with MG132 for 3 h and then stimulated with anti-CD3 antibody for 15 min. The FLAG-SLP-76 proteins were immunoprecipitated and immunoblotted with anti-ubiquitin (Lys-48) antibody. D, measurement of the degradation of Ser-376-phosphorylated SLP-76 (WT and K30R mutant) by cycloheximide chase assay. J14 cells were stimulated with anti-CD3 antibody for 15 min and then treated with 50 μg/ml cycloheximide (CHX) for 0–120 min. The cell lysates were collected, and the levels of the indicated proteins were analyzed by Western blotting. Data are representative of three independent experiments. E, simple linear regression analysis of the relative Ser-376-phosphorylated SLP-76 protein levels in D at different times of cycloheximide treatment. The relative Ser-376-phosphorylated SLP-76 protein levels were quantified and normalized to β-actin protein levels. The equation of the protein degradation rate for the Ser-376-phosphorylated WT SLP-76 protein in J14 cells is as follows: Y = −0.005032*X + 0.9911 (R2 = 0.8167, p = 0.0001). The equation of the protein degradation rate for the Ser-376-phosphorylated SLP-76 K30R mutant protein in J14 cells is as follows: Y = −0.001609*X + 0.9671 (R2 = 0.6136, p = 0.0074).