Figure 4
Figure 4. caSTAT5a, but not WT STAT5a, downregulates the expression of LEF-1 protein, an effect that is rescued by bortezomib. (A) (Top) HEK293 cells were transfected with an LEF-1 expression plasmid with our without co-transfection of caSTAT5a or WT STAT5a. Whole-cell lysates were analyzed for LEF-1 and β-actin protein expression by western blotting. All samples were processed in a similar fashion, as described in Materials and Methods. Representative images are shown. (Bottom) Bar graphs indicate the ratio between the optical density of protein bands of LEF-1 protein to that of the housekeeping protein, β-actin. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05). (B) (Top) HEK293 cells were transfected with an LEF-1 expression plasmid, with our without co-transfection of caSTAT5a, and subsequently treated with 1 μg/μL of bortezomib (BZ) or dimethylsulfoxide. Whole-cell lysates were analyzed for LEF-1 and β-actin protein expression by western blotting. All samples were processed in a similar fashion, as described in Materials and Methods. Representative images are shown. (Bottom) Bar graphs indicate the ratio between the optical density of protein bands of LEF-1 protein to that of the housekeeping protein, β-actin. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05). (C) HEK293 cells were transfected with an LEF-1 expression plasmid, with our without co-transfection of caSTAT5a or WT STAT5a, and subsequently treated with 1 μg/μL BZ. Lysates of transfected cells were immunoprecipitated with a polyclonal anti–LEF-1 antibody and the immunoprecipitates were subjected to western blot analysis with a monoclonal anti–LEF-1 antibody. The IgG antibody band was used as a loading control. Representative western blot images are shown. (D) LEF-1 mRNA expression in HEK293 cells transfected as described in panel C. mRNA was measured by qRT-PCR. LEF-1 mRNA levels were normalized to those of β-actin and are expressed as AUs. Data represent means ± SD and are derived from 2 independent experiments, each in triplicate (*P < .05, **P < .01). (E) HEK293 cells were transfected with an LEF-1 expression plasmid, with or without co-transfection of caSTAT5a or WT STAT5a, and subsequently incubated with 1 μg/mL of BZ alone or in combination with 1 μg/mL of cycloheximide (CHX). After 24 hours, mRNA was isolated and LEF-1 mRNA expression was measured by qRT-PCR. LEF-1 mRNA levels were normalized to those of β-actin and are presented as AUs. Data represent means ± SD and are derived from 2 independent experiments, each in duplicate (*P < .05, **P < .01).

caSTAT5a, but not WT STAT5a, downregulates the expression of LEF-1 protein, an effect that is rescued by bortezomib. (A) (Top) HEK293 cells were transfected with an LEF-1 expression plasmid with our without co-transfection of caSTAT5a or WT STAT5a. Whole-cell lysates were analyzed for LEF-1 and β-actin protein expression by western blotting. All samples were processed in a similar fashion, as described in Materials and Methods. Representative images are shown. (Bottom) Bar graphs indicate the ratio between the optical density of protein bands of LEF-1 protein to that of the housekeeping protein, β-actin. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05). (B) (Top) HEK293 cells were transfected with an LEF-1 expression plasmid, with our without co-transfection of caSTAT5a, and subsequently treated with 1 μg/μL of bortezomib (BZ) or dimethylsulfoxide. Whole-cell lysates were analyzed for LEF-1 and β-actin protein expression by western blotting. All samples were processed in a similar fashion, as described in Materials and Methods. Representative images are shown. (Bottom) Bar graphs indicate the ratio between the optical density of protein bands of LEF-1 protein to that of the housekeeping protein, β-actin. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05). (C) HEK293 cells were transfected with an LEF-1 expression plasmid, with our without co-transfection of caSTAT5a or WT STAT5a, and subsequently treated with 1 μg/μL BZ. Lysates of transfected cells were immunoprecipitated with a polyclonal anti–LEF-1 antibody and the immunoprecipitates were subjected to western blot analysis with a monoclonal anti–LEF-1 antibody. The IgG antibody band was used as a loading control. Representative western blot images are shown. (D) LEF-1 mRNA expression in HEK293 cells transfected as described in panel C. mRNA was measured by qRT-PCR. LEF-1 mRNA levels were normalized to those of β-actin and are expressed as AUs. Data represent means ± SD and are derived from 2 independent experiments, each in triplicate (*P < .05, **P < .01). (E) HEK293 cells were transfected with an LEF-1 expression plasmid, with or without co-transfection of caSTAT5a or WT STAT5a, and subsequently incubated with 1 μg/mL of BZ alone or in combination with 1 μg/mL of cycloheximide (CHX). After 24 hours, mRNA was isolated and LEF-1 mRNA expression was measured by qRT-PCR. LEF-1 mRNA levels were normalized to those of β-actin and are presented as AUs. Data represent means ± SD and are derived from 2 independent experiments, each in duplicate (*P < .05, **P < .01).

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