Translocation of CD74-ICD to the nucleus is required for TAp63 transcription. (A) HEK-293 cells were transfected for 20 hours with various constructs of CD74. Transfection efficiency was determined by analyzing the mRNA levels of a sequence that appears in all CD74 constructs. RNA was isolated and TAp63 transcription was determined by RT-PCR. The results presented are representative of 3 different experiments. (B) HEK-293 cells were transfected with empty vector or a construct containing CD74-ICD conjugated to a nuclear localization signal (1–42 nuc). Total RNA was extracted and real-time PCR was performed as described in “Real-time reverse-transciption–PCR analysis.” mRNA levels are shown after normalization for the HPRT control. Error bars represent SD. (C,D) HEK-293 cells were transfected with empty vector or a construct containing CD74-ICD conjugated to a nuclear localization signal (1-42 nuc). TAp63α and γ transcription was followed by RT-PCR. The results presented are representative of 8 independent experiments (C). Western blot showing protein expression of p63 isotypes. Cells were collected and lysed as described in “Cell lysis by hot SDS,” and lysates were separated on a 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. The results presented are representative of 5 different experiments (D). The intensity of the p63 band was calculated as described in Figure 2.