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. 2013 Jul 1;210(7):1331–1350. doi: 10.1084/jem.20120812

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© 2013 Jackstadt et al.

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Figure 2. — Validation of AP4-mediated differential gene expression. (A and B) The induction (A) or repression (B) of selected genes was confirmed by qPCR analysis with mRNA isolated at the indicated time points from DLD-1 cells infected with an adenovirus expressing AP4. Detection of β-actin was used for normalization. Results represent the mean ± SD (n = 3), and in the case of FOS mean ± SE (n = 2). (C) Expression of ectopic and endogenous AP4 was determined by immunoblot analysis using an AP4-specific antibody. Lysates were obtained from SW620 or DLD-1/pRTR-AP4-VSV cells treated with DOX for 24 h. (D) Lysates from DLD-1/pRTR-AP4-VSV cells treated with DOX for the indicated periods were subjected to immunoblot analysis for the indicated proteins. (E) Quantification of mRNA expression of several putative AP4 target genes after activation of a conditional AP4 allele in U2OS cells by addition of DOX for the indicated periods. Expression was normalized to β-actin expression. Experiments were performed in triplicates. Results represent the mean ± SD (n = 3). (F) Detection of the indicated protein expression levels by immunoblot analysis after activation of a conditional AP4 allele in U2OS cells by addition of DOX for the indicated periods. (G) Detection of the indicated protein expression levels by immunoblot analysis after activation of a conditional AP4 allele in SW480 cells by addition of DOX for the indicated periods. In C, D, F and G, the detection of α-tubulin served as a loading control.