Bα subunit of PP2A interacts with EBP50.

<p>Panels <i>A</i> and <i>B</i>: Bacterially expressed glutathione S-transferase (GST) and GST-tagged wild-type EBP50 were loaded onto glutathione-Sepharose as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035595#s4" target="_blank">Materials and Methods</a>. After a washing step the resin samples were incubated with BPAEC lysate or cell lysis buffer. Non-binding proteins were washed out and the bound proteins were eluted with 10 mM glutathion. Coomassie staining <i>(A)</i> and Western blot probed with PP2A A, B, B′, or C specific antibodies <i>(B)</i> of the bacterial and endothelial cell lysates (Total) and the eluted fractions after the pull-down are shown. Panel <i>C</i>: BPAEC monolayers were transfected with pcDNA3.1 V5-His (vector ctr), pcDNA3.1 V5-His PP2A Bα (PP2A Bα–V5) and pcDNA3.1 V5-His B′γ (PP2A B′γ–V5) plasmids. Lysates of the transfected cells were incubated with Anti-V5 Agarose Affinity Gel as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035595#s4" target="_blank">Materials and Methods</a>, and the bound proteins were eluted by boiling the resin in 1× SDS sample buffer. Western blot analysis of the lysates of transfected cells (Total) and eluted samples were done using EBP50 (upper panel) and V5-tag (lower panel) specific antibodies. Additional bands at 55 kD are IgG. Representative data of three independent experiments are shown.