Chlamydial hsp60

The cells were seeded on cover slips and infected with Chlamydia trachomatis serovar L 2 at MOI 1 for indicated time points. Before fixation with 4% PFA/Sucrose (Roth), the cells were washed with DPBS (Gibco TM ). Fixed cells were permeabilized with 0.2% Triton-X-100 (Sigma-Aldrich) in 1x DPBS for 30 min, blocked with 2% FBS in 1x DPBS for 45 min and incubated with primary antibodies for 1 h at room temperature. Primary antibodies were diluted in 2% FBS in 1x DPBS; chlamydial HSP60 (Santa Cruz, 1:500) and Phalloidin (Thermo Fischer). Samples were washed and incubated with fluorescence dye conjugated secondary antibodies (Dianova) for 1 h in the dark at room temperature. Cover slips were mounted onto microscopy slides using mowiol, slides were air dried for at least 24 h and examined using a LEICA DM2500 fluorescence microscope. The images were analysed with LAS AF program and Image J software.

For Western blot analysis, cells were directly lysed with 2x Laemmli (10% 1.5M Tris-HCl pH 6.8, 4% SDS, 30% glycerol and 1.5% ß-mercaptoethanol) buffer on ice. Protein samples were separated with a 10% SDS-PAGE (Peqlab) and transferred onto a PVDF membrane (Sigma-Aldrich) via a semi-dry blotter (Peqlab) (2 hours at 1 mA per cm 2 ). The membrane was blocked in 5% (w/v) non-fat dried milk powder in 1x Tris-buffered saline with 0.5% Tween20 (Sigma-Aldrich) for 1 h and then incubated in the appropriate primary antibody over night at 4°C; c-Myc, anti c-Myc phospho-T58 and anti c-Myc phospho-S62 were purchased from Abcam. Phospho-Akt (Ser473), Akt (pan), phospho-Erk, Erk, phospho-GSK-3ß (Ser9), (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. GSK-3ß (3D10), phospho-Stat1 (Ser727), phospho-Stat1 (Tyr701) and Stat1 were obtained from Cell Signaling. ß-Actin was purchased from Sigma-Aldrich. Chlamydial HSP60 was obtained from Santa Cruz and OmpA was self-made. Proteins were detected with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz), using homemade ECL solutions and Intas Chemiluminescence Imager.