FIG. 2.
Effect of HasA(1-174)E148A, D167A on HasA secretion. Cultures of E. coli MC4100 with plasmids pSyc150 (HasDE), pAM-HasA, or pAM-HasAE148A, D167A and pTrc99A-HasA(1-174) or pTrc99A-HasA(1-174)E148A, D167A were grown and induced with 0.5 mM IPTG. After 3 h of induction, culture aliquots were centrifuged. Proteins in the supernatants were precipitated with trichloroacetic acid, separated by 14% SDS-PAGE, and visualized by Coomassie blue staining (top panel). Proteins present in the cell pellets were electrotransferred to a nitrocellulose membrane and immunoblotted with anti-HasA polyclonal antiserum (bottom panel). Loaded samples were normalized to an OD600 of 0.2. The molecular masses of the markers (kilodaltons) are indicated at the left.