Methods T as the activity T betr Gt 2.1. Reagents. Culture media and additives were tze purchased from Sigma, unless Geldanamycin otherwise indicated. PK mouse anti-mouse anti-p85 PI3K pSer473Akt rabbit anti-mouse KK Body Antiphosphotyrosinantik 4G10, rabbit Y182 T180 antiphospholipid p38, p38 p44 phospho T202 Y204 42, 42 p44, mouse anti-GAPDH, rabbit anti ? I BC 15 and Goat anti-actin HRP goat anti-mouse HRP-anti-mouse IgG rabbit. LY294002 LY303511 Bay were obtained from Calbiochem 11 and 7085. Inhibitors of PI3K isoform-specific in vitro studies were provided by Kevan Shokat. TGX 221 and IP 103 Mice were in recombination experiments acquired Chemdea LPS of E. coli H18 flagellin ver Ffentlicht described in. The human IL-8 and IL-6 mouse ELISA R & D Systems.
Flagellin S Ttigungskonzentrationen proximal pathways in Caco 2 and 100 ng ml In experiments with the PI3K inhibitors or shRNA that this dose, the upper limit for the production of IL-8 2.2 is used. Cell culture. Caco 2 cells were obtained from MLN8237 the American Type Culture Collection and grown inDMEMwith LD 4.5 g of glucose, amino acids 1x nonessential, 2 mM glutamine, penicillin and streptomycin at 10 f Tales K K. K Calf serum Caco 2 cells were cultured in a saturated density of 106 ml of t plated tt 5 experiences and removed 14 days after confluence. HEK 293T cells were obtained from ATCC and are S Ure S in DMEM with 10 FBS S Acids by heat, penicillin, streptomycin, cultural and non-essential amino Acids inactivated. T84 cells from ATCC were cultured in DMEM F12 with 15 mM HEPES, streptococci, and pin 5 FBS.
T in 5105, they were sown per well and t 3 t used 4 days after sowing. 2.3. Immunopr zipitation and Western blot. Flagellin treatment of cells in 12-well plates or 6 were different times by two washes with phosphate buffer-L Solution followed Salzl IceCold culture. Treated cells were then lysed in 250 l of lysis buffer and equal volumes of 500 protein lysate from each sample by Western blot analyzes. For Zipitation Immunpr of PI3K, p85 5 g to 500 g of the cell lysate at 4 bench ? ?C night. Immune complexes were bound to protein A-agarose for 1 hour, washed in lysis buffer and analyzed by Western blotting using 4G10 to 1: 1000. 2.4. IL-8 promoter report. Caco 2 cells with pEGFP and IL-8 promoter-luciferase reporter, and 96-well plates were inoculated as T stimulated in.
After six days described by electroporation cells and lysed in Bright Glo reagent. Married Ratio ratio Ratio ratio Ratio ratio Ratio of luminescence in arbitrary units of fluorescence was calculated for each sample and multiply this value refers to the same embroidered experience is as a Erh Increase both the expression defined. 2.5. Determination of PI3K kinase in vitro. Caco 2 postconfluence for at least 5 days of culture 6-well plates. They were serum overnight and then with flagellin points starve in a short time. The cells were lysed with cold PBS, and the fight against p85 and protein A-agarose was washed. In the second washing stage 20, the total volume is collected each R Hrchen and analyzed by Western blot with all examined p85. Immunopr Zipitaten remaining were subjected to in vitro kinase