CD74 up-regulates TAp63 α and γ expression. (A) HEK-293 cells were transfected with GFP or CD74-GFP constructs for 20 hours. Total RNA was isolated and reverse transcription was carried out using Superscript II RT. RT-PCR was performed to determine p63, TAp63, ΔNP63, TAp63α, and TAp63γ mRNA levels. The results presented are representative of 3 to 10 experiments. (B) HEK-293 cells were transfected with GFP or CD74-GFP constructs for 20 hours. Cells were collected and lysed as described in “Cell lysis by hot SDS,” and lysates were separated on 5% to 15% (wt/vol) gradient SDS-PAGE and blotted with anti-p63 antibody followed by HRP-conjugated antirabbit antibodies. The arrows indicate bands of 80 and 56 kDa, representing p63TAα and p63TAγ. The results presented are representative of at least 4 separate experiments. The intensity of the p63 band was divided by the intensity of the actin or tubulin band. The activation fold ratio in the GFP-transfected cells was normalized to 1, and the ratio for each transcription was calculated as the intensity of the sample, relative to 1.