Rhogdi

Proteins were extracted in RIPA buffer (50 mM Tris-base, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) and quantified by the DC protein assay kit (Bio-Rad). Samples were separated by 12% SDS-PAGE and transferred to PVDF membranes (Amersham Biosciences, Piscataway, NJ, USA). After blocking with 5% non-fat milk in Tris-buffered saline, 0.1% Tween 20 for 1 h, the membranes was incubated overnight at 4 °C with respective primary antibodies. The primary antibodies for RhoGDI, JNK, p-JNK, Akt/p-Akt and MAPK/p-MAPK were purchased from Cell Signaling Technology (Beverly, MA, USA). The primary antibodies for c-Jun, p-c-Jun, caspase-3 and GAPDH were acquired from Santa Cruz Biotechnology. After that, the blots were incubated with secondary antibody conjugated to HRP for 2 h at room temperature. Target proteins were detected by enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

RAW 264.7 cells were plated at a density of 1 × 10⁷ cells per well in 10-cm dishes and then challenged with L. pneumophila-GFP strains at an MOI of 50. When appropriate, cells were incubated for 1 h before Legionella challenge with 50 µg/mL CHX or 10 µM MG132. At 2 hpi, cells were washed with 1× PBS solution and harvested. Cells were fluorescently sorted based on GFP signal by using a BD INFLUX sorter. Postsorted infected cells (GFP⁺) and bystander cells (GFP⁻) were centrifuged and lysed by using 2× SDS Laemmli sample buffer (0.125 M Tris⋅HCl, pH 6.8, 4% SDS, 20% glycerol, 20% glycerol, 10% β-mercaptoethanol, 0.01% bromophenol blue) and boiled for 10 min. Proteins were transferred to PVDF membranes, blocked in 5% BSA, and probed with antibodies to cyclin D1 (Abcam), cyclin E1 (Cell Signaling), RhoGDI (Cell Signaling), GAPDH (Cell Signaling), or tubulin (Sigma). Immunoblotting with primary antibodies was carried out overnight at 4°. Dylight anti-rabbit IgG 680 and anti-mouse IgG 800 were used as secondary antibodies (1:20,000; Cell Signaling). Capture and analysis of membranes was performed by using an Odyssey scanner and Image Studio software (LI-COR Biosciences).