A pressure of 100–350 hPa was used to deliver 1–2 pl of suspension per pulse. Approximately one bacterium was successfully delivered into a cell every two pulses. Following nanoblade delivery, cells find more were washed twice with HBSS before the addition of fresh medium with 250 μg/mL kanamycin [24, 26]. Immunoprecipitation HEK293T cells were first seeded in a 6 well plate at a density of 1 x 106 cells per well and then infected with the required strain the
following day. At required time points, cells were lysed with lysis buffer (50 mM Tris pH 7.5, 0.1 mM EGTA, 0.27 M sucrose, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 1% Triton-100, protease inhibitor cocktail). Protein G sepharose beads (Sigma-Aldrich) were pre-incubated with total TAK1 antibody (kind
gift from Dr. Peter Cheung, Nanyang Technological University, Singapore) before the cell lysates were mixed and incubated with the beads for 1 hr. at 4°C with shaking. Beads were then washed twice with lysis buffer and twice with wash buffer (50 mM Tris–HCl pH 7.5, 0.27 M Sucrose, 0.1% 2-mercaptoethanol) before being boiled in SDS-PAGE sample https://www.selleckchem.com/products/cilengitide-emd-121974-nsc-707544.html buffer. Samples were subsequently resolved on SDS-PAGE gels and transferred onto nitrocellulose membrane (Pall Life Sciences). Western blotting Cells were lysed with MPer mammalian protein Selleckchem KPT-8602 extraction reagent (Thermo Scientific) supplemented with protease cocktail (Thermo Scientific). Proteins were then quantitated using Bradford reagent (Bio-Rad). Samples were boiled Acetophenone in SDS-PAGE sample buffer and 50 μg (per lane) were resolved on an SDS-PAGE gel and transferred onto nitrocellulose membranes (Pall Life Sciences). The membranes were then blocked with 5% BSA at room temperature for 1 hr. and probed with specific antibodies at 4°C overnight followed by secondary antibody anti-rabbit IgG, HRP-linked for 1 hr. at room temperature. Antibodies were obtained from Cell Signaling Technology except the β
-actin antibody (Sigma-Aldrich). Blots were developed on film (Pierce Chemical) using ECL plus Western blotting substrate (Thermo Scientific). Statistical analysis NFκB reporter assays were performed in triplicates. Results were presented as mean ± standard deviation. Student’s t-test was used to find the significant differences between the means. The significant differences were reported as p < 0.05 (*) and p < 0.01 (**). Acknowledgments We thank Mark P Stevens (Institute of Animal Health, UK) for the BopE and SopE expression plasmids and Peter Cheung (Nanyang Technological University) and Liu Xinyu (NUS) for technical advice on the TAK1 immunoprecipitations and Western blots.
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