Analysis of localization and secretion of expressed proteins in flagellar mutant strains. (A) Western blot analyses with anti-Peb1 and anti-flagella antibodies of whole cells and cell-free culture supernatants from the strains indicated above the panels and expressing wt Peb1 (from pLA25) and FliC1-183 peptide (from pLA31), respectively. Note secretion of Peb1 in MKS111ΔHBB but lack of FliC1-183 secretion in the same strain. (B) Subcellular localization of Peb1 expressed from a plasmid or from the chromosome. Top panel shows SDS-PAGE and middle panel Western blot analysis with anti-Peb1 antibodies of whole -cell samples and periplasmic extracts of MKS12 (pLA25), MKS12P
peb1, MKS12 (pMCS3'UTR), and MKS12. Lack of cell lysis and cytoplasmic leakage was controlled by immunoblotting with anti-RNAP antibodies (bottom panel). (C) SDS-PAGE (top panel) and Western blot (middle panel) analyses of whole cell, cell-free culture supernatant and periplasmic samples of strains MKS12 and MKS111ΔHBB carrying the plasmids pLA25 (encodes Peb1) and pLA32 (encodes FliC1-20Peb1). Lack of cytoplasmic leakage was controlled by immunoblotting with anti-RNAP antibodies (bottom panel). In all the panels of the figure, the letter C indicates whole-cell samples, S cell-free culture supernatants, and PP periplasmic extracts. All the samples correspond to 100 μl of culture and molecular weight markers in kDa are indicated on the left.