Coexpressed ephrin-A3 attenuates EphA receptor activation in cancer cells.

<p>(A,B) NCI-H226 and A549 lung cancer cells were infected with a lentivirus encoding EphA3 and ZsGreen alone or together with a lentivirus encoding mCherry-ephrin-A3; control cells were infected with lentiviruses encoding ZsGreen and mCherry. EphA3 immunoprecipitates were probed by immunoblotting for phosphotyrosine (PTyr) and reprobed for EphA3. Lysates were probed for mCherry-ephrin-A3 with an anti-dsRed antibody that also recognizes mCherry, for EphA3, and for β-tubulin as loading control. The histograms show normalized means ± SE quantified from 3 immunoblots in both A and B. In one of the A549 experiments used for quantification, the cells were stimulated with ephrin-A5 Fc. **p<0.01 by one sample t test for the comparison of ephrin-A3 Fc-treated cells expressing both EphA3 and ephrin-A3 with ephrin-A3 Fc-treated cells expressing only EphA3. Of note, EphA3 levels were higher in A549 cells co-expressing ephrin-A3/ephrin-B2 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081445#pone-0081445-g002" target="_blank">Figs. 2A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081445#pone-0081445-g003" target="_blank">3B</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081445#pone-0081445-g004" target="_blank">4A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081445#pone-0081445-g005" target="_blank">5C,D</a>), suggesting that this receptor may be stabilized by the coexpressed ephrins. (C) A549 cells were infected with a lentivirus encoding mCherry-ephrin-A3 or mCherry as a control. Immunoprecipitated endogenous EphA2 was probed by immunoblotting for phosphotyrosine (PTyr) and reprobed for EphA2. Lysates were probed with an anti-dsRed antibody and β-tubulin as loading control. The histogram shows normalized means ± SE quantified from 3 immunoblots. **p<0.01 by one sample t test for the comparison of ephrin-A3 Fc-treated cells expressing or not expressing ephrin-A3.