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. Author manuscript; available in PMC: 2020 May 2.
Published in final edited form as: Sci Signal. 2019 Apr 16;12(577):eaar3349. doi: 10.1126/scisignal.aar3349

Fig. 3. The PKCθ C2 domain interacts with the kinase Zap70.

Fig. 3.

(A) Purified recombinant GST-tagged WT or HR2A mutant forms of the PKCθ C2 domain, GST-C2 and GST-C2-HR2A, respectively, were used for pull-down assays of lysates from unstimulated or PV-stimulated Jurkat T cells. Bound material was separated by SDS-PAGE and immunoblotted for phosphotyrosine (pTyr) and GST. The boxed area was subjected to LC-MS/MS analysis. Data are representative of 3 independent experiments.

(B) Identity of some of the peptides resolved by LC-MS/MS from PV-stimulated Jurkat T cells.

(C) Representative pull-down experiment using purified recombinant GST-C2 and GST-C2-HR2A to pull down proteins from lysates of unstimulated or PV-stimulated primary CD4+ T cells from PKCθ knockout mice. Pulled-down material (top panels) or whole-cell lysates (WCL) were analyzed by immunoblotting for the indicated proteins. Data are representative of 3 independent experiments.

(D–G) Primary B6 (D and E) or OT-II (F and G) CD4+ T cells were left unstimulated or stimulated for the indicated times with BM-derived dendritic cells (APC) pulsed with SEE or OVA peptide, respectively. PKCθ immunoprecipitates (IP) or WCL were immunoblotted for the indicated proteins. Representative immunoblots of 3 independent experiments are shown (D and F) alongside quantification of Zap70 by densitometry (E and G) pooled from 3 independent experiments. *P < 0.05, **P < 0.01.