Mouse carotid artery ligation The carotid artery ligation model of remodeling was performed after buprenorphine analgesia and induction of anesthesia using inhalational isoflurane essentially as described deubiquitination assay previously and conformed with all the Guide for the Care and Use of Laboratory Animals. All procedures were approved by the University of Rochester Animal Care Committee. Immunohistochemistry and histomorphometry Mice were perfusion fixed with 10 % paraformaldehyde in sodium phosphate buffer, fourteen days after ligation. A number of cross-sections were made from the bifurcation every 200 lm by way of a 2 mm length of carotid artery and stained with either hematoxylin and eosin and antibodies against an actin, PCNA, Bax, Hrt 1 and GSK 3b, as described previously. Media pressure was calculated from media tension/h, where h is medial width, decided histomorphometrically. Immunocytochemistry SMC were seeded onto 6 well plates 2 times Neuroblastoma before being stained at 2 9 105 cells per well. Cells were stained for phospho GSK 3b, Notch3 or Notch1 at 80 90% confluency utilising the following process. Cells were washed 3 times in 19 PBS. The cells were fixed and then permeabilized in methanol, and subsequently re-hydrated in 19 PBS/3% BSA. Cells were then incubated in the right primary antibody at 4 C over night with gentle agitation. Following three 10 min washes in 19 PBS, cells were incubated in the appropriate secondary antibody for 2 3 h at 37 C. Cells were then washed once in 19 PBS before visualization with the use of an Olympus DP 50 fluorescent microscope, using appropriate excitation and emission spectra at 209 magnifications. GSK 3b showing vectors The wild type GSK 3b expression plasmid and the constitutively active mutant GSK 3b S9A, where the serine from position 9 is changed by an alanine, were kind gifts of Dr. Jim Woodgett of the Samuel Lenfeld c-Met Inhibitor Research Institute, Toronto, Canada. As described previously, plasmids were prepared for transfection based on the manufacturers instructions utilizing a Qiagen plasmid midi system. Plasmid preparation, transient transfection, luciferase and w galactosidase assays Plasmids were prepared for transfection according to the manufacturers instructions as described previously using a Qiagen plasmid midi kit. The cells were transfected with a luciferase reporter construct, and different expression constructs. Transfection efficiency was established and normalized to w galactosidase task following corp transfection with pCMV LacZ. Western blot analysis was also performed to ensure over expression of effector proteins. Cells were collected 16 24 h post transfection, using 19 Reporter Lysis Buffer. Transactivation of reporter genes was normalized to the t galactosidase activity and examined from the luciferase assay. The latter was conducted in line with the manufacturers guidelines.