Figure 7.
Figure 7. Signaling pathways of Jurkat cells leading to activation of AP-1, NF-κB, and NFAT and to induced tyrosine phosphorylation are dependent on MAL expression. (A) The three types of Jurkat cell were conjugated to APCs pulsed with SEE for the indicated times. Cell extracts were analyzed by immunoblotting to detect the activated, phosphorylated (p) forms or the total content of Erk, p38, and JNK as indicated (left). Cells were transfected with a luciferase reporter plasmid whose transcription was controlled by AP-1. Cells were then conjugated to APCs, which were pulsed or not with SEE. After 4 h, luciferase activity was measured (right). (B) The three types of Jurkat cell were conjugated for the indicated times to APCs pulsed with SEE. Cell extracts were analyzed by immunoblotting to detect the activated phosphorylated forms of IKK-α/β or the total content of IKK-α (left). Cells were transfected with a luciferase reporter plasmid whose transcription was controlled by NF-κB. Cells were then conjugated to APCs, which were pulsed or not with SEE. After 4 h, luciferase activity was measured (right). (C) Cells were conjugated to APCs pulsed with SEE for 1 h at 37°C. Cell extracts were analyzed by immunoblotting with anti-NFAT antibodies that detect both the inactive phosphorylated NFAT and the active dephosphorylated NFAT forms. Control of cells treated for 1 h with PMA plus ionomycin (Io) in the absence or presence of cyclosporin A (CsA) was included to help identification of phosphorylated and dephosphorylated NFAT (left). The three types of Jurkat cell were transfected with a luciferase reporter plasmid whose transcription was controlled by NFAT. Cells were then conjugated to APCs, which were pulsed or not with SEE. After 4 h, luciferase activity was measured (right). (D) Cells were treated or not with activating anti-CD3 UCHT1 antibodies for 15 min. Cell extracts were immunoprecipitated with antiphosphotyrosine PY20 mAb coupled to agarose. The phosphotyrosine immunoprecipitates were immunoblotted to detect tyrosine phosphorylated CD3ζ, ZAP-70, and PLC-γ1 (top). The original extracts were immunoblotted to show the total content of CD3ζ, ZAP-70, and PLC-γ1 (bottom). (E) The three types of Jurkat cell were transiently transfected with plasmid DNA containing the luciferase gene under the control of the IL-2 promoter. After 16 h, cells were conjugated to APCs, which were pulsed or not with SEE (top), or incubated or not with PMA plus ionomycin (Io; bottom). After 4 h, cell extracts were used to determine luciferase activity. Data are represented as mean ± SEM of the luciferase activity in stimulated cells relative to that in unstimulated cells.

Signaling pathways of Jurkat cells leading to activation of AP-1, NF-κB, and NFAT and to induced tyrosine phosphorylation are dependent on MAL expression. (A) The three types of Jurkat cell were conjugated to APCs pulsed with SEE for the indicated times. Cell extracts were analyzed by immunoblotting to detect the activated, phosphorylated (p) forms or the total content of Erk, p38, and JNK as indicated (left). Cells were transfected with a luciferase reporter plasmid whose transcription was controlled by AP-1. Cells were then conjugated to APCs, which were pulsed or not with SEE. After 4 h, luciferase activity was measured (right). (B) The three types of Jurkat cell were conjugated for the indicated times to APCs pulsed with SEE. Cell extracts were analyzed by immunoblotting to detect the activated phosphorylated forms of IKK-α/β or the total content of IKK-α (left). Cells were transfected with a luciferase reporter plasmid whose transcription was controlled by NF-κB. Cells were then conjugated to APCs, which were pulsed or not with SEE. After 4 h, luciferase activity was measured (right). (C) Cells were conjugated to APCs pulsed with SEE for 1 h at 37°C. Cell extracts were analyzed by immunoblotting with anti-NFAT antibodies that detect both the inactive phosphorylated NFAT and the active dephosphorylated NFAT forms. Control of cells treated for 1 h with PMA plus ionomycin (Io) in the absence or presence of cyclosporin A (CsA) was included to help identification of phosphorylated and dephosphorylated NFAT (left). The three types of Jurkat cell were transfected with a luciferase reporter plasmid whose transcription was controlled by NFAT. Cells were then conjugated to APCs, which were pulsed or not with SEE. After 4 h, luciferase activity was measured (right). (D) Cells were treated or not with activating anti-CD3 UCHT1 antibodies for 15 min. Cell extracts were immunoprecipitated with antiphosphotyrosine PY20 mAb coupled to agarose. The phosphotyrosine immunoprecipitates were immunoblotted to detect tyrosine phosphorylated CD3ζ, ZAP-70, and PLC-γ1 (top). The original extracts were immunoblotted to show the total content of CD3ζ, ZAP-70, and PLC-γ1 (bottom). (E) The three types of Jurkat cell were transiently transfected with plasmid DNA containing the luciferase gene under the control of the IL-2 promoter. After 16 h, cells were conjugated to APCs, which were pulsed or not with SEE (top), or incubated or not with PMA plus ionomycin (Io; bottom). After 4 h, cell extracts were used to determine luciferase activity. Data are represented as mean ± SEM of the luciferase activity in stimulated cells relative to that in unstimulated cells.

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