Figure 5
Figure 5. CD74 induces activation of the p65 member of the NF-κB family, which in turn activates TAp63 transcription. HEK-293 cells were transfected with empty, p65, or p65 + p50 constructs for 20 hours. (A) Analysis of HEK-293 cells transfected with NF-κB proteins by electrophoresis mobility shift assay using the NF-κB–binding site as DNA probe. (B) Nuclear extracts from p65-, p65 + p50–, and p50-transfected cells were used for DNA-binding reactions as indicated at the top the lanes. Total RNA was isolated, and RT-PCR was performed as described in “RNA isolation and reverse transciption.” (C) Cells were then collected and lysed, as described in “Cell lysis by hot SDS.” Lysates were separated on 5% to 15% (wt/vol) gradient SDS-PAGE, and blotted with anti-p63 antibody, followed by HRP-conjugated antirabbit antibodies. Arrows indicate bands of 80 and 56 kDa, representing p63TAα and p63TAγ. (D) Schematic representation of the p63 promoters. The sequences indicated are the PCR primers used for generating the luciferase constructs. (E,F) TAp63 activation was analyzed by luciferase assay, as described in “Luciferase assay for monitoring p63 activation.” (E) The HEK-293 cells were transfected with a luciferase construct containing the TAp63 promoter, together with a p65 construct or CD74-ICD (aa's 1-42), in the absence or presence of IκB. Cells were lysed, and luciferase activity was measured. Luciferase activities were normalized to the activity of the cotransfected RSV promoter–driven Renilla reporter luciferase, which was used to correct for differences in transfection efficiencies. Fold activation was calculated as the activity of p65 or CD74 constructs relative to the activity of the empty plasmid. The results presented are representative of 3 different experiments. (F) HEK-293 cells were transfected for 20 hours with luciferase constructs containing the TAp63 or ΔNp63 promoters, together with CD74-ICD (aa 1-42's). Cells were then lysed and luciferase activities were normalized to the activity of cotransfected RSV promoter–driven Renilla reporter luciferase, which was used to correct for differences in transfection efficiencies. Fold activation was calculated as the activity of the CD74-ICD construct, relative to the activity of cells transfected with an empty plasmid. The graph represents the average of 5 independent experiments. The results presented are representative of 3 different experiments. The intensity of the p63 band was calculated as described in Figure 2. Error bars represent SD.

CD74 induces activation of the p65 member of the NF-κB family, which in turn activates TAp63 transcription. HEK-293 cells were transfected with empty, p65, or p65 + p50 constructs for 20 hours. (A) Analysis of HEK-293 cells transfected with NF-κB proteins by electrophoresis mobility shift assay using the NF-κB–binding site as DNA probe. (B) Nuclear extracts from p65-, p65 + p50–, and p50-transfected cells were used for DNA-binding reactions as indicated at the top the lanes. Total RNA was isolated, and RT-PCR was performed as described in “RNA isolation and reverse transciption.” (C) Cells were then collected and lysed, as described in “Cell lysis by hot SDS.” Lysates were separated on 5% to 15% (wt/vol) gradient SDS-PAGE, and blotted with anti-p63 antibody, followed by HRP-conjugated antirabbit antibodies. Arrows indicate bands of 80 and 56 kDa, representing p63TAα and p63TAγ. (D) Schematic representation of the p63 promoters. The sequences indicated are the PCR primers used for generating the luciferase constructs. (E,F) TAp63 activation was analyzed by luciferase assay, as described in “Luciferase assay for monitoring p63 activation.” (E) The HEK-293 cells were transfected with a luciferase construct containing the TAp63 promoter, together with a p65 construct or CD74-ICD (aa's 1-42), in the absence or presence of IκB. Cells were lysed, and luciferase activity was measured. Luciferase activities were normalized to the activity of the cotransfected RSV promoter–driven Renilla reporter luciferase, which was used to correct for differences in transfection efficiencies. Fold activation was calculated as the activity of p65 or CD74 constructs relative to the activity of the empty plasmid. The results presented are representative of 3 different experiments. (F) HEK-293 cells were transfected for 20 hours with luciferase constructs containing the TAp63 or ΔNp63 promoters, together with CD74-ICD (aa 1-42's). Cells were then lysed and luciferase activities were normalized to the activity of cotransfected RSV promoter–driven Renilla reporter luciferase, which was used to correct for differences in transfection efficiencies. Fold activation was calculated as the activity of the CD74-ICD construct, relative to the activity of cells transfected with an empty plasmid. The graph represents the average of 5 independent experiments. The results presented are representative of 3 different experiments. The intensity of the p63 band was calculated as described in Figure 2. Error bars represent SD.

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