FIG. 1.

Membrane association of LidA after translocation into host cells. (A) L. pneumophila (WT) and an isogenic dotA mutant (dotA), both expressing GFP, were grown to postexponential phase and resuspended in PBS containing either 1% Triton X-100, 1% CHAPS, 1% OG, or 1% digitonin (see Materials and Methods). The detergent-soluble and -insoluble fractions were analyzed by immunoblotting using antibodies directed against LidA or GFP. (B) U937 cells were incubated for 1, 6, or 12 h with either L. pneumophila (WT) or the isogenic dotA mutant (dotA), both expressing GFP, prior to incubation for 20 min in PBS containing 1% digitonin. The detergent-soluble and -insoluble fractions were analyzed by immunoblotting using antibodies directed against LidA or GFP. Lp, wild-type L. pneumophila grown in bacteriological medium and directly resuspended in sample buffer; Uninf., uninfected. (C) U937 cells were incubated for 6 h with either L. pneumophila (WT) or the isogenic dotA mutant (dotA), both expressing GFP, prior to mechanical fractionation (see Materials and Methods). The cytosolic fraction was separated from the membrane fraction by ultracentrifugation, and the membrane fraction was subjected to digitonin extraction (see Materials and Methods). The cytosolic fraction (Cytoplasm) and the digitonin-soluble fraction (Membrane Dig. Sol.) were analyzed by immunoblotting using antibodies directed against LidA, calnexin, or RhoGDI.