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. Author manuscript; available in PMC: 2014 Jan 24.
Published in final edited form as: Mol Cancer Res. 2010 Sep 21;8(10):1388–1398. doi: 10.1158/1541-7786.MCR-10-0042

FIGURE 5.

FIGURE 5

Effect of MDM2 expression on ErbB4/Kap1/p53 interactions. A and B, ErbB4 and Kap1. HEK293T cells were infected with pSiren or pSiren shMDM2 virus. After 24 h, the cells were transfected with pcDNA3.1 V5/His or s80 Cyt2 in the same vector. At 72 h after infection, the cells were harvested and total protein was extracted. A, immunoblots for endogenous MDM2, Kap1, and GAPDH and ectopic s80 ErbB4 in cell lysates. B, anti-V5 immunoprecipitation, with ErbB4 or Kap1 immunoblot from the samples analyzed in A. C, ErbB4, Kap1, and p53. COS-7 cells were transfected with vector (V; lanes 1 and 5); with 2 µg of truncated CYT-1 ErbB4 and 2 µg of Flag-tagged Kap1 (EK; lanes 2 and 6); with2 µg of truncated CYT-1 ErbB4, 2 µg of Flag-tagged Kap1, and 2 µg of MDM2 (EKM; lanes 3 and 7); and with 2 µg of truncated CYT-1 ErbB4, 2 µg of Flag-tagged Kap1, and 4 µg of MDM2 (EKM; lanes 4 and 8). Cells were treated with 10 µmol/L MG132 for 3.5 h and stimulated with TPA for 30 min before harvest at 48 h. Data represent two biological replicates in identical format plus two similar biological replicates (A and B).