EDTA, and the fluorescence intensity of the cells was analyzed using a FACScan flow cytometer at 518 nm e citation and 605 nm emission for DHE and at 495 nm e citation and 529 nm emission for DCF. Determination of NADPH o idase activity http://www.selleckchem.com/products/crenolanib-cp-868596.html by chemiluminescence assay After incubation with LPS, cells were gently scraped Inhibitors,Modulators,Libraries and centrifuged at 400 g for 10 min at 4 C. The cell pellet was resuspended with 35 ul per well of ice cold RPMI 1640 medium, and the cell suspension was kept on ice. To a final 200 ul volume of pre warmed RPMI 1640 medium containing Inhibitors,Modulators,Libraries either NADPH or lucigenin, 5 ul of cell suspension was added to initiate the reaction followed by immediate measure ment of chemiluminescence in an Appliskan luminometer in an out of coincidence mode. Appropriate blanks and controls were established, and chemilumines cence was recorded.
Inhibitors,Modulators,Libraries Neither NADPH nor NADH enhanced the background chemiluminescence of lucigenin alone. Chemiluminescence was continuously measured for 12 min, and the activity of NADPH o idase was e pressed as counts per million cells. Western blot analysis Growth arrested cells were incubated with LPS at 37 C for the indicated time intervals. The cells were washed, scraped, collected, and centrifuged at 45000 g at 4 C for 1 h to yield the whole cell e tract, as previously described. Samples were denatured, subjected to SDS PAGE using a 12% running gel, and transferred to nitrocellulose membrane. Membranes were incubated with an anti VCAM 1 antibody for 24 h, and then incubated with an anti mouse horseradish pero idase antibody for 1 h.
The immunoreactive bands were detected by ECL reagents. RT PCR analysis Total RNA was isolated with Trizol according to the protocol of the manufacturer. The cDNA obtained from 0. 5 ug total RNA was used as a template for PCR amplification as previously described. Real time RT PCR analysis Inhibitors,Modulators,Libraries Total RNA was e tracted using TRIzol reagent. mRNA was reverse transcribed into cDNA and analyzed by real time RT PCR. Real time PCR was performed using SYBR Green PCR reagents and primers specific for VCAM 1 and GAPDH mRNAs. The levels of VCAM 1 e pression were deter mined by normalizing to GAPDH e pression. Transient transfection with siRNAs The small interfering RNA duple es correspond ing to human No 2, No 4, TLR2, TLR4, MyD88, p47pho , c Src, p38 MAPK, ATF2, and p300 and scrambled siRNA were from Invitrogen.
Transient transfec tion of siRNAs was carried out using Metafectene trans fection reagent from Bionte Lab. siRNA was formulated with Metafectene transfection reagent according to the manufacturers instruction. Cilengitide Isolation of cell fractions Cells were harvested, sonicated for 5 s at output 1. 5 with a sonicator, and centri fuged at 8000 rpm for 15 min at 4 C. The pellet was col lected as the nuclear fraction. The supernatant was centrifuged at 14000 rpm at 4 C for 60 min to yield the pellet and the supernatant. Measurement Ivacaftor mw of VCAM 1 luciferase activity For construction of the VCAM 1 luc plasmid, human VCAM 1 promoter,