jejuni. EGF was added to cells five min just before inoculation with C. jejuni. Quantification of membrane ruffling was finished by two independent ob servers and tabulated. Only cells with clear boundaries were counted and cells favourable for membrane ruffling had been scored. Adenylate cyclase domain reporter delivery assays The delivery with the C. jejuni CiaC protein to HeLa cells was determined employing laboratory established protocols. We made use of the adenylate cyclase domain of the Bordetella pertussis CyaA protein fused to your C. jejuni CiaC protein as being a reporter for protein delivery to host cells. ACD catalyzes the production of cAMP from ATP only inside the presence in the host cell protein calmodulin. As being a negative control, the metK gene driven from its native promoter was also cloned in frame with the ACD.
The C. jejuni MetK protein is often a cytosolic S adenosylmethionine synthetase. Briefly, HeLa cells were pre treated with MBCD, noco dazole, cytochalasin D, TAE 226, and DMSO for 30 minutes before inoculation with C. jejuni. The amount of intra cellular cAMP created in HeLa cells was assayed at thirty min post infection by selleck chemical ELISA. The assay was repeated five occasions to make certain reproducibility. Assessment of Rho GTPase Rac1 activation HeLa cells have been seeded into six nicely tissue culture plates at a density of two ? 105 cells properly and serum starved for 24 hours. Cells had been pre treated with motor vehicle, MBCD, nocodazole, and cytochalasin D for 30 minutes just before inoculation with C. jejuni resuspended in PBS. The amount of activated Rac1 in C.
jejuni infected and uninfected cells was de termined using the G LISA Rac1 Activation Assay ac cording towards the manufacturers instructions. Transfection of cells with smaller interfering RNA or plasmids HeLa cells had been transfected with tiny interfering RNA utilizing lipofectamine RNAiMax selleck inhibitor in accordance on the manufacturers directions. Caveolin one stealth siRNA and scrambled control siRNA have been applied towards the cells 24 h just before the assay. A conventional binding and internalization assay was then carried out. Knockdown of endogenous proteins was confirmed by immunoblot with antibodies for specific host proteins. Caco 2 cells were transfected with plasmids en coding for caveolin one eGFP or the empty vector manage eGFP applying Lipofectamine LTX and Plus Reagent accord ing to producers instructions HeLa cells have been seeded at three ? 106 cells properly and serum starved in MEM for three h just before the addition of C. jejuni with and devoid of therapy also as an uninfected. Cells had been pretreated 30 min before and throughout C. jejuni infection with both ten ug ml PP2, twenty uM erlotinib, and or five uM TAE 226, or with 5 mM MBCD additional at the time of infection. At 45 min following infec tion, cells have been collected in ice cold lysis buffer.