Figure 3.

The SepL/SepD/CesL complex does not participate in the calcium‐absence response. (a) Immunoblot analysis of whole‐cell lysates of the EPEC sepL‐3FLAG cesL‐2HA strain grown under T3S inducing conditions in the presence or absence of calcium. Intracellular protein stability of SepL‐3FLAG and CesL‐2HA was examined every 30 min during 3 hr after the addition of chloramphenicol, by immunodetection using anti‐FLAG and anti‐HA antibodies. Anti‐DnaK was used as a loading control. (b) Immunoprecipitation of SepL‐3FLAG from the EPEC sepL‐3FLAG cesL‐2HA and EPEC cesL‐2HA strains grown in the presence or absence of calcium. Coimmunoprecipitation of SepD, CesL‐2HA, and DnaK with SepL‐3FLAG was corroborated with specific antibodies against SepD, HA‐tag, and DnaK. (c) The EPEC sepL‐3FLAG cesL‐2HA strain grown in the presence or absence of calcium was fractionated into cytoplasmic (C) and membrane (M) fractions. Immunodetection of SepL‐3FLAG and CesL‐2HA in the cell lysate (Lys) and in the cytoplasmic and membrane fractions was performed using antibodies against the FLAG and HA tags. To confirm proper fractionation, samples were probed with anti‐DnaK and anti‐EscJ antibodies as cytoplasmic and membrane markers, respectively