Immunoblot analysis of sub-cellular localization of PrtV protein in V. cholerae.

(A) Immunoblot analyses of cell fractions from V. cholerae wild type strain C6706 (lanes 1, 3, 5, and 7) and the prtV mutant (lanes 2, 4, 6, and 8) using anti-PrtV serum (upper panel), anti-Crp antiserum (middle panel), and anti-β-lactamase antiserum (lower panel). Lanes 1 and 2: whole cell lysates; lanes 3 and 4, cytoplasmic fractions; lanes 5 and 6, periplasmic fractions; lanes 7 and 8, culture supernatants. Asterisks indicate the 81 kDa PrtV protein (B) Proteinase K susceptibility assay. OMVs from V. cholerae wild type strain C6706 were treated with 0.5 μg ml-1 of proteinase K (PK), 1% SDS and/or the proteinase K inhibitor PMSF (1 mM) as indicated. Samples were examined by immunoblot analysis using polyclonal anti-PrtV antiserum (upper panel). Lane 1: OMVs treated with only PK; lane 2: OMVs treated with SDS and PK; lane 3: OMVs treated with SDS, PMSF, and PK; lane 4: control OMVs. The same membrane was re-probed with OmpU antiserum as an internal control (lower panel).