Anti tubulin

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 earlier^{52 (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 (OD_{600 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-MavC^{26 (link)}, 1:5000; anti-tubulin (Developmental Studies Hybridoma Bank, E7) 1:10,000; anti-ICDH^{53 (link)}, 1:10,000.

Mixed stage worms were lysed and solubilized in SDS lysis buffer (2% SDS, 100 mM NaCl, 10% glycerol, and 50 mM Tris HCl, pH 6.8) with sonication. Worm extracts were separated on SDS-PAGE and transferred onto PVDF membranes. The separated proteins were probed with anti-GFP (Cell Signaling Technology, Danvers, MA, #2956, RRID:AB_10828931) or anti-tubulin (Developmental Studies Hybridoma Bank, AA4.3, RRID:AB_579793) antibodies. Pixel intensities of GFP and tubulin bands were quantified using Adobe Photoshop CC 2015 (RRID:SCR_014199).