Figure 5.
Figure 5. Homer-3 regulates SRE activation in Jurkat cells. (A) Jurkat cells were electroporated with SRE luciferase reporter (1 μg), GFP construct (0.2 μg), and Myc construct either empty or expressing full-length Homer-3 (5, 10, or 15 μg). Twenty hours later the cells were stimulated with OKT3 (5 μg/mL) or PMA (50 ng/mL) for an additional 4 hours. Luciferase activity was determined as described in “Materials and methods.” Overexpression of Homer-3 reduced SRE activation induced by OKT3 and especially by PMA. Three independent experiments were performed with duplicate samples. Histograms represent the mean ± SE (A-B, D-E). (B) Jurkat cells were electroporated with SRE luciferase reporter (1 μg), GFP construct (0.2 μg), and Myc construct either empty or expressing each fragment of Homer-3 (15 μg). Twenty hours later the cells were stimulated with PMA (50 ng/mL) for an additional 4 hours. Luciferase activity was determined as described in “Materials and methods.” The expression of each fragment was confirmed with Western blotting. Overexpression of the EVH1 fragment reduced SRE activation induced by PMA, although the expression level of EVH1 fragment was much less than that of the other fragments. (C) Jurkat cells were electroporated with constructs expressing SiRNA directed against Homer-3 (no. 1, nucleotides 218-236 of coding sequence; no. 2, nucleotides 138-156; 10 μg). After 72 hours' incubation, the cells were lysed and blotted for endogenous Homer-3 expression. Only SiRNA construct of Homer-3 no. 2 suppressed Homer-3 expression, whereas expression level of Vav1 and actin was not affected. (D) To assess SRE activation under knockdown of Homer-3, Jurkat cells were electroporated with SRE luciferase reporter (2 μg), GFP construct (0.2 μg), and SiRNA constructs (10 μg). After 68 hours' incubation, the cells were stimulated with PMA (50 ng/mL) for an additional 4 hours. Luciferase activity was determined as described in “Materials and methods.” SRE activation was elevated only after electroporation of SiRNA construct of Homer-3 no. 2. (E) Jurkat cells were electroporated with SRE luciferase reporter (1 μg), GFP-Ha-RasG12V, or empty construct (2.5 μg) and Myc construct either empty or expressing each fragment of Homer-3 (15 μg). Twenty-four hours later, luciferase activity was determined as described in “Materials and methods.” Overexpression of full-length or EVH1 domain fragments of Homer-3 reduced SRE activation induced by Ha-RasG12V. Immunoblotting confirmed expression of GFP-Ha-RasG14V and each fragment of Homer-3 (data not shown).

Homer-3 regulates SRE activation in Jurkat cells. (A) Jurkat cells were electroporated with SRE luciferase reporter (1 μg), GFP construct (0.2 μg), and Myc construct either empty or expressing full-length Homer-3 (5, 10, or 15 μg). Twenty hours later the cells were stimulated with OKT3 (5 μg/mL) or PMA (50 ng/mL) for an additional 4 hours. Luciferase activity was determined as described in “Materials and methods.” Overexpression of Homer-3 reduced SRE activation induced by OKT3 and especially by PMA. Three independent experiments were performed with duplicate samples. Histograms represent the mean ± SE (A-B, D-E). (B) Jurkat cells were electroporated with SRE luciferase reporter (1 μg), GFP construct (0.2 μg), and Myc construct either empty or expressing each fragment of Homer-3 (15 μg). Twenty hours later the cells were stimulated with PMA (50 ng/mL) for an additional 4 hours. Luciferase activity was determined as described in “Materials and methods.” The expression of each fragment was confirmed with Western blotting. Overexpression of the EVH1 fragment reduced SRE activation induced by PMA, although the expression level of EVH1 fragment was much less than that of the other fragments. (C) Jurkat cells were electroporated with constructs expressing SiRNA directed against Homer-3 (no. 1, nucleotides 218-236 of coding sequence; no. 2, nucleotides 138-156; 10 μg). After 72 hours' incubation, the cells were lysed and blotted for endogenous Homer-3 expression. Only SiRNA construct of Homer-3 no. 2 suppressed Homer-3 expression, whereas expression level of Vav1 and actin was not affected. (D) To assess SRE activation under knockdown of Homer-3, Jurkat cells were electroporated with SRE luciferase reporter (2 μg), GFP construct (0.2 μg), and SiRNA constructs (10 μg). After 68 hours' incubation, the cells were stimulated with PMA (50 ng/mL) for an additional 4 hours. Luciferase activity was determined as described in “Materials and methods.” SRE activation was elevated only after electroporation of SiRNA construct of Homer-3 no. 2. (E) Jurkat cells were electroporated with SRE luciferase reporter (1 μg), GFP-Ha-RasG12V, or empty construct (2.5 μg) and Myc construct either empty or expressing each fragment of Homer-3 (15 μg). Twenty-four hours later, luciferase activity was determined as described in “Materials and methods.” Overexpression of full-length or EVH1 domain fragments of Homer-3 reduced SRE activation induced by Ha-RasG12V. Immunoblotting confirmed expression of GFP-Ha-RasG14V and each fragment of Homer-3 (data not shown).

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