Resolved proteins were detected by their sellckchem specific primary antibodies and horseradish peroxidase conjugated second ary antisera. The immunoblots were visualized by chemilu minescence with the ECL kit from Amersham, and the images detected in X ray films were quantified by densito metric scanning using the Eagle Eye II still video system. Measurement of intracellular Ca2 by FLIPR The intracellular Ca2 was measured by using an opti mized Fluorometric Imaging Plate Reader proto col. HeLa cells were seeded into clear bottomed black walled 96 well plates. The growth medium was re placed by 200 uL labeling medium containing 1 1 ATCC MEM medium Hanks balanced salt solution, 2. 5% fetal calf serum, 20 mmol L HEPES, pH 7. 4, 2. 5 mmol L probenecid and 2 umol L Fluo 4 AM.

Hista mine was prepared as a 5 solution in Hanks balanced salt solution into another polypropylene Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries 96 well plate. After 1 h labeling, cell and drug plates were placed in a FLIPR. Imme diately after the addition of 50 uL of drug solution into the cell medium, changes in fluorescence were monitored over 120 s following excitation at a wavelength of 488 nm and detection at 510 560 nm. Luciferase assay The growth medium of serum starved transfectants was removed and replaced by 25 ul of lysis buffer provided in the Luciferase Reporter Gene Assay kit. The 96 well microplate was shaken on ice for 30 min. The luciferase activity was de termined by a microplate luminometer LB96V. Injector M connected to lysis buffer and injector P connected to the luciferin substrate were set to inject 25 ul of each component into each well.

A 1. 6 sec delay time followed by a 2 sec measuring time period was assigned to infector M whereas injector P was measured for 10 s after introduction of luciferin into the well. Results were collected by WinGlow version 1. 24 and expressed as relative luminescent units. Statistical calculation was performed using KyPlot Inhibitors,Modulators,Libraries version 2. 0. Establishment of stable cell lines HEK293 or H1299 cells stably expressing Flag tagged Fhit, or the pFlag CMV2 vector were established by LipofectAMINE mediated transfection along with ex cess pcDNA3, followed by G418 selection for 2 weeks. The resultant cell lines were named as 293 Fhit and 293 vector, or H1299 Fhit and H1299 Inhibitors,Modulators,Libraries vector, respectively. 3 2,5 dephenyl tetrazolium bromide colorimetric assay Cells were seeded in 96 well plates and incubated in the absence or presence of agonists for various durations.

After re moving the growth medium, 100 ul MTT labeling re agent Inhibitors,Modulators,Libraries in serum free medium was added. The plate was incubated for 4 h at 37 C prior to the addition of 100 ul solubilization buffer. The plate was incubated over night at this research 37 C. The absorbance reading was taken at the wavelength of 570 nm, with the reference value taken at the wavelength of 630 nm.