Methods Cell line Osteoblasts were maintained in Dulbeccos modified Eagles medium supplemented with 10% heat inactivated FTY720 order FBS, 2 mM glutamine and 100 units/ml penicillin/streptomycin at 37 C in a humidified atmos phere of 5% CO2 in air. DQ gelatin degradation assay Coverslips were coated with 100 ug/ml quenched fluores cence substrate DQ gelatin. MG 63 cells were incubated with 100 ug/ml EMD protein in the presence or absence of tissue inhibitor of metalloprotein ases 2 for 20 h, followed by incubating on DQ gelatin coated plates for a period of 4 h. Cells were fixed with 2% paraformaldehyde in PBS. Slides were mounted with coverslips using glycerol/PBS, and examined with at 488 nm and 533 nm using an Olympus LSM GB200 equipped with an oil immersion lens.

Differential interference contrast was used to visualize cells cultured on the matrix. Western blot analysis MG 63 cells were preincubated with 100 ng/ml 5 uM SB203580 for 30 min at 37 C, and MG 63 cells were then placed in serum free DMEM with 100 ug/ml EMD protein for 48 h. Conditioned media were collected, centrifuged to remove debris, and concentrated in Amicon Centriprep concen trators up to 10 fold. Cells were incubated in serum free Eagle medium with 100 ug/ml EMD protein for 48 h. MG 63 cells prepared as described above were lysed with SDS sample buffer and sonicated briefly in order to shear DNA. Samples were separated on 10% SDS polyacrylamide gels under reducing conditions. Proteins were electrophoretically transferred to polyvinylidene difluoride mem branes.

Membranes were incubated for 1 h with anti phospho p38 antibody or anti p38 antibody in PBS containing 0. 05% Tween 20 and 10% Blockace. Peroxidase conjugated secondary anti body was used at a 1 1,000 dilution and immunoreactive bands were visualized using Super Signal west pico chemiluminescent substrate. Signals on each membrane were analyzed by VersaDoc 5000. Reverse transcription polymerase chain reaction Total RNA was isolated from MG 63 cells cells by RNeasy kit. MG 63 cells were then placed in serum free DMEM with 100 ug/ml EMD protein for 12 h. After denaturation of total RNA at 70 C for 10 min, cDNA was synthesized with oligo dT primer by incubating with reverse transcriptase at 50 C for 30 min. Polymerase chain reactions were PCR products were loaded to agarose gel, and stained with ethidium bromide. The bands were analyzed using AlphaImager IS 3400 software. Briefly, IDV was measured as the sum of all the pixel values after background correction in each band. The values of each band were calculated as IND/AREA, where AREA is the size of the region that was measured. The results are shown the values of AVGMMP1/AVGGAPDH Dacomitinib at each time point.