Figure 7
Figure 7. Binding of CMS to the pTα cytoplasmic tail is involved in pre-TCR signaling. (A) Expression levels of pre-TCR (TCRβ-pTα) heterodimers and pTα monomers in JR3.11 cells transduced with Flag-tagged versions of either pTαWT or pTαΔPro1 were detected by anti-Flag immunoprecipitation, followed by nonreducing SDS-PAGE, and Western blotting with an anti-Flag mAb. Endogenous CMS expression was detected by inmmunoblotting with anti-CMS. (B) Surface expression levels of pre-TCR complexes on pTαWT and pTαΔPro1 JR3.11 GFP+-transduced cells were analyzed by flow cytometry with an anti-CD3ϵ mAb. (C) Time-course response in calcium mobilization obtained from the indicated cell lines after stimulation with an anti-CD3ϵ mAb. Ionophore treatment was used to control for cell viability and intact calcium stores. Data are represented as the FL4/FL5 ratio of emission analyzed by flow cytometry at the indicated times. Data are representative of at least 4 independent experiments. (D) Deletion of the Pro1 motif (ΔPro1) of the human pTα tail impairs pre-TCR–induced NFAT transcriptional activity. pTαWT and pTαΔPro1 JR3.11 cells transiently transfected with an NFAT–luciferase-reporter plasmid were stimulated with an anti-pTα mAb or left unstimulated. NFAT transcriptional activity is expressed as the fold increase of luciferase activity above levels obtained from the nonstimulated control in a representative experiment (left), or as the mean (± SEM) of relative NFAT activity of 6 independent experiments (right); *P < .005. (E) Western blot analysis of CMS expression in pTαWT and pTαΔPro1 JR3.11 cells. Total lysates of cells transfected with an empty vector, or with a vector encoding either full-length CMS or a CMS truncated form encompassing the SH3ABC CMS domains, were analyzed by immnubloting with anti-CMS or anti-Flag. (F) CMS modulates preTCR-induced NFAT transcriptional activity. pΤαWT and pTαΔPro1 JR3.11 cells were transfected with either an empty vector, or a vector encoding either full-length CMS or a CMS truncated form encompassing the SH3ABC CMS domains. NFAT transcriptional activity was analyzed upon stimulation with anti-pTα as the mean (± SEM) of relative NFAT activity of 4 independent experiments. *P < .001 and **P < .05.

Binding of CMS to the pTα cytoplasmic tail is involved in pre-TCR signaling. (A) Expression levels of pre-TCR (TCRβ-pTα) heterodimers and pTα monomers in JR3.11 cells transduced with Flag-tagged versions of either pTαWT or pTαΔPro1 were detected by anti-Flag immunoprecipitation, followed by nonreducing SDS-PAGE, and Western blotting with an anti-Flag mAb. Endogenous CMS expression was detected by inmmunoblotting with anti-CMS. (B) Surface expression levels of pre-TCR complexes on pTαWT and pTαΔPro1 JR3.11 GFP+-transduced cells were analyzed by flow cytometry with an anti-CD3ϵ mAb. (C) Time-course response in calcium mobilization obtained from the indicated cell lines after stimulation with an anti-CD3ϵ mAb. Ionophore treatment was used to control for cell viability and intact calcium stores. Data are represented as the FL4/FL5 ratio of emission analyzed by flow cytometry at the indicated times. Data are representative of at least 4 independent experiments. (D) Deletion of the Pro1 motif (ΔPro1) of the human pTα tail impairs pre-TCR–induced NFAT transcriptional activity. pTαWT and pTαΔPro1 JR3.11 cells transiently transfected with an NFAT–luciferase-reporter plasmid were stimulated with an anti-pTα mAb or left unstimulated. NFAT transcriptional activity is expressed as the fold increase of luciferase activity above levels obtained from the nonstimulated control in a representative experiment (left), or as the mean (± SEM) of relative NFAT activity of 6 independent experiments (right); *P < .005. (E) Western blot analysis of CMS expression in pTαWT and pTαΔPro1 JR3.11 cells. Total lysates of cells transfected with an empty vector, or with a vector encoding either full-length CMS or a CMS truncated form encompassing the SH3ABC CMS domains, were analyzed by immnubloting with anti-CMS or anti-Flag. (F) CMS modulates preTCR-induced NFAT transcriptional activity. pΤαWT and pTαΔPro1 JR3.11 cells were transfected with either an empty vector, or a vector encoding either full-length CMS or a CMS truncated form encompassing the SH3ABC CMS domains. NFAT transcriptional activity was analyzed upon stimulation with anti-pTα as the mean (± SEM) of relative NFAT activity of 4 independent experiments. *P < .001 and **P < .05.

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