Legionella strains used in this study were derivatives of Philadelphia 1 strain Lp02 and were grown and maintained on charcoal-yeast extract (CYE) plates or in ACES-buffered yeast extract (AYE) broth as described earlier52 (link). Genes were inserted into pZL507 (ref. 53 (link)) for complementation experiments. Raw264.7 cells and U937 cells were purchased from ATCC were cultured in RPMI1640 medium supplemented with 10% FBS.
For infection experiments, all L. pneumophila strains were grown overnight in AYE broth to postexponential phase judged by the optical density of the cultures (OD600 nm = 3.2–3.8) and by increase in bacterial motility, then 0.2 mM IPTG was added into bacterial cultures to induce the expression of MavC and its mutants on pZL507 at 37 °C for 3 h before infection. Raw264.7 cells or differentiated U937 macrophages were infected with an multiplicity of infection (MOI) of 15 at 37 °C for 2 h. Infected cells washed three times with PBS were lysed with 0.2% saponin for 30 min on ice. Saponin-soluble fractions were probed with UBE2N and MavC-specific antibodies for the modified UBE2N and the translocated MavC, respectively.
Antibodies: Anti-UBE2N (Thermo Fisher Scientific, cat. no. 37-1100), 1:1000; anti-MavC26 (link), 1:5000; anti-tubulin (Developmental Studies Hybridoma Bank, E7) 1:10,000; anti-ICDH53 (link), 1:10,000.
For infection experiments, all L. pneumophila strains were grown overnight in AYE broth to postexponential phase judged by the optical density of the cultures (OD600 nm = 3.2–3.8) and by increase in bacterial motility, then 0.2 mM IPTG was added into bacterial cultures to induce the expression of MavC and its mutants on pZL507 at 37 °C for 3 h before infection. Raw264.7 cells or differentiated U937 macrophages were infected with an multiplicity of infection (MOI) of 15 at 37 °C for 2 h. Infected cells washed three times with PBS were lysed with 0.2% saponin for 30 min on ice. Saponin-soluble fractions were probed with UBE2N and MavC-specific antibodies for the modified UBE2N and the translocated MavC, respectively.
Antibodies: Anti-UBE2N (Thermo Fisher Scientific, cat. no. 37-1100), 1:1000; anti-MavC26 (link), 1:5000; anti-tubulin (Developmental Studies Hybridoma Bank, E7) 1:10,000; anti-ICDH53 (link), 1:10,000.
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