Figure 4. Mutation of the s80^HER4 D-box eliminates mitotic degradation of s80^HER4 and enhances s80^HER4-mediated growth inhibition. A.

Mutation of the s80^HER4 D-box stabilized s80^HER4 during mitosis. A.1 Western analysis of HER4 IP's from HeLa-s80, -s80^db, and -s80^KD cells cultured 24 h +TET. IP's were analyzed for phospho-tyrosine or GFP. A.2: HeLa-s80^db cells were cultured +NOC/+TET in 10% serum (16 h). Cells were released into 10% serum +TET/+CHX at T=0, and extracts collected at 15 min intervals. Expression of s80^db and cyclin B was analyzed as described in Fig. 3. B. Equal numbers of cells were plated at day 0 ±TET. Cells were counted at 1-day intervals through 4 days. P= 0.011 comparing cell number of HeLa-s80^HER4 +TET at day 4 to cell number of HeLa-s80^db + TET at day 4, n=5, analyzed in triplicate. C. Cell cycle analysis of HeLa-s80^db cells cultured ±TET (48h). > 10,000 nuclei were analyzed per condition; n=3; representative histograms shown. D. Cells were in 10% serum +HU/+TET for 16 h to synchronize in S-phase. Cells were released into 10% serum +TET at T=0 h. Cells were collected at 0, 2, 4, 8, 12, and 24 h following HU release. Cells were stained with propidium iodide and analyzed by flow cytometry as described above.