B. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT) cells before washing cells (before wash) and after washing and growth (after wash) in absence (NT) or presence (bulgecin) of bulgecin A. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis) and HcpHA were immunodetected using anti-TolB (upper panel) and anti-HA (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown. 