SctA is massively associated with secreted pycnosomes.

<p>(A) The SctA and B proteins were expressed in bacteria as GST-fusion proteins, purified, and analyzed by Western blot with anti-GST and B4.2 antibodies. While an anti-GST antibody labeled both proteins, the B4.2 antibody only recognized GST-SctA and not SctB. (B) Supernatants (S6, S15, S100) and corresponding pellets (P15, P100) obtained by differential centrifugation of <i>D</i>. <i>discoideum</i> cell culture medium (600, 15’000, 100’000 x <i>g</i>, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154875#pone.0154875.g002" target="_blank">Fig 2</a>) were analyzed by Western blot using the B4.2 antibody. Equal volumes were loaded for all samples. (C) <i>D</i>. <i>discoideum</i> cells were cultured for 4 days. Cells and a fraction enriched in secreted pycnosomes were recovered by successive centrifugation of cell suspension at 600 x <i>g</i> and 100’000 x <i>g</i> respectively. Serial two-fold dilutions from the two fractions were analyzed by Western blot using the B4.2 antibody, the H161 anti-p80 and an antibody against mitochondrial porin. The number of cells loaded on each lane is indicated above each band. The fraction of each protein associated to secreted pycnosomes is indicated on the right.