Journal: Nature Communications
Article Title: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17
Figure Lengend Snippet: Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), PMSF (1 mM) or MG132 (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
Article Snippet: Chemicals and antibodies Chemicals were obtained from the following sources: Hoechst 33342 (Thermo Fisher Scientific), PMSF (Wako chemicals), MG132 (Calbiochem), TRAIL (Merck Millipore), STS (Wako chemicals) and Percoll (Sigma-Aldrich).
Techniques: Transfection, Plasmid Preparation, Fractionation, Mass Spectrometry, Incubation, Staining