In addition, ionizing radiation induced phosphorylation of 992 and 1173 while betacellulin induced 1068 (Sturla et al., 2005; Saito et al., 2004). ERK. Incubation in the presence of a functional obstructing antibody to HB-EGF also resulted in a decrease in the phosphorylation of EGFR. In addition, cell migration was inhibited by CRM197 and rescued when cells were incubated with HB-EGF. We showed that injury induced phosphorylation of specific ZL0454 tyrosine residues and found that a similar pattern of phosphorylation was induced by trinucleotides. These studies show that injury induced purinergic receptor activation prospects to phosphorylation of EGFR, ERK and migration. test. 2.7 Measurement of ATP Released with Injury The release of ATP after injury was assayed using a luciferin-luciferase bioluminescence assay according to the protocol of Molecular Probes. The assay is based on the requirement of ATP for luciferase to produce light. Cells were cultured to confluency in P-35’s and the medium was replaced with 250 l HEPES. Cultures were wounded and 10 l of the medium was removed immediately after injury and measured using a luminometer (Luminoskan Ascent 2.5, Thermo Lab Systems Waltham, MA). The ATP released into the medium of hurt cells was compared to control cells (medium change only). Experiments were performed five occasions with an internal replicate of three each time. 2.8 Immunoprecipitation Cells were cultured to confluency, washed with PBS, placed on ice and lysed in 10 mM Tris-HCl (pH 7.4) containing 0.1% SDS, 1% ZL0454 Triton X-100, 1% deoxycholate, 5 mM ethylenediamine-tetraacetic acid (EDTA), 2 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium orthovanadate (Na3VO4), 1g/ml aprotinin, 1g/ml leupeptin and 1g/ml pepstatin. The lysates were centrifuged at 10,000g for quarter-hour at 4C. The supernatant was precleared with protein A beads and mouse IgG, and the primary antibody was added to the supernatant in the concentration of 5g/ml, followed by over night incubation at 4C. Washed protein A slurry was added to the supernatant and rocked at 4C for 4 hours. The combination was centrifuged at 3,000g for 30 mere seconds at 4C, and the pellet was washed with lysis buffer and prepared for SDS-PAGE. The producing phosphorylation was normalized to total EGFR. 2.9 SDS PAGE and Western Blot Analysis Lysates were collected and sheared as explained previously (Yang et al., 2004). The protein concentration of the supernatant was identified using the BCA assay. Comparative amounts of protein from each lysate (40 g) were Icam1 subjected to SDS-PAGE and transferred to polyscreen PVDF membrane (PerkinElmer, Boston, MA). Blots were blocked inside a Tris buffer (10 mM Tris, 100 mM NaCl, 0.1% Tween-20) containing 0.2% I-block (Applied Biosystems, Foster City, CA) and membranes were incubated with appropriate primary antibodies, washed and incubated with appropriate secondary antibodies and rinsed with TBST. Immunodot blot assays were performed within the lysates collected from the 2 2 well slides and probed for pERK and ERK. Visualization was performed by enhanced chemiluminescence (PerkinElmer, Boston MA) and quantified with the Kodak Imaging system. Responses were normalized to control. 2.10 Immunohistochemical Analysis Cells were cultivated to confluency and either stimulated with EGF or scrape wounded. A parallel group of cells were incubated with TIMP-3 for one hour prior to activation. After 5 min, cells were rinsed with PBS, ZL0454 fixed for 20 min with 3.7% formalin (pH 7.2). Cells were prepared for immunohistochemical staining as explained previously (Klepeis et al., 2004). Cells were clogged with 5% BSA/PBS and then incubated over night at 4C with the antibody of interest in 3% BSA/PBS. Cells were rinsed with PBS, clogged and incubated with Alexa 488 anti-mouse IgG (Invitrogen, Eugene OR). Bad controls were incubated without the primary antibody. Cells were imaged and analyzed.