Polarity lost PTEN SHIP1 adversely Chtigten chemotaxis PtdInsP3 Zelladh recession Type SHIP1-/-Wild band 23 1 April 2012 in SHIP1 Zelladh sion and migration | 1229 test mission Zelladh 96-well MP-470 plates with 10 g μ / ml fibronectin at 37 ° C for 1 h and blocked with 1% BSA in phosphate-buffered salt solutions coated solution for 1 h bone marrow neutrophils were isolated, resuspended at a density of 1 � �� � 07/ml, and either left unstimulated or stimulated with fMLP min for 2. Bone marrow neutrophils were then added to each well and keep for 5, 15 or 30 minutes. Adherent cells were removed by washing three times with PBS. The cells were lysed with 20 l μ 0.5% hexadecyltrimethylammonium bromide. Peroxidase activity t was then reflected in cell lysates using TMB as a substrate to the amount of cells present.
The reaction was min between 10 and 15, and the absorbance was read at 450 nm adjusted. VX-680 Total input was used as a contr To Zelladh Sion compared to determine for comparison. Immunpr Were zipitation neutrophils from the bone marrow either with fMLP for 2 min, and stimulated to lie keep it on a surface surface coated with fibronectin for 30 min. Stimulated neutrophils in suspension were cases than controls in both cases. Neutrophils IP lysis buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100 and 2 mM EDTA with inhibitors erg Complements protease. The cell lysates were mixed with SHIP1 Antique Incubated body, and immune complexes were drawn with protein A + G agarose beads. Pull eluates were resolved by SDS AGE � St �� and probed for SHIP1 and tyrosine-phosphorylated proteins using 4G10.
The chemotaxis assay taxi MIC-1000 EZ-Scan was used to examine real-time horizontal chemotaxis of neutrophils M Mice. The EZ-Scan, a substrate GE taxi Tzten silicon and a plate of flat glass to form both the two chambers. Glass slides hunters, which were coated with 0.1% and 2% BSA or 10 μ g / ml fibronectin arranged on the glass plate in the battery compartment. Purified bone marrow � �� erived wild-type and SHIP1 � � �� � �n eutrophils in HBSS/Ca2 + + Mg 2 + 0.1% BSA, 2% BSA, 0.1% BSA or with 1 μ g / ml RGD peptide were chtigt with the lower container lter given every six canals and erm le, box 18 by removing the buffer μ the upper reservoir. An amount of 1 l chemotactic μ was added to the upper reservoir.
Chemotaxis was stirred at 37 ° C for 20 at 30 s intervals using a charge-coupled-device camera. Were followed for the analysis of traces of cells, neutrophils migrate coordinates from sequential images with imaging software DIAS. Cell tracks were then so that all cells have been started from the same starting point and were plotted using Matlab GE changed. Direktionalit t as the course in a straight line from the origin through the compl Length of migration is shared. The migration rate was calculated as the average velocity of the cells for each captured image. The parameters were only for cell migration during the w calculated 5 – to 15-minute period of each film. SHIP1 phosphatase Total 5 � �� � 06 Neutrophils were either in suspension or on fibronectin-coated surface Che lysed in IP lysis buffer and pulled down using SHIP1 SHIP1 Antique Body and protein A + G agarose beads.
The beads were washed three times with phosphatase reaction buffer containing 25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2.7 mM KCl and 10 mM dithiothreitol washed. SHIP1 was folded with 3000 pmol of substrate PtdInsP3 L Soluble in water, incubated for 30 � 0 min at 37 �� C produced ° Without phosphate from SHIP1 activity t was then quantified by the malachite green phosphatase assay kit according to claim manufacturer’s protocol. PtdInsP3 percent conversion was determined for each time that � �� � 00% / 3000 pmol. Without phosphate in the background, the value of phosphate in � is �s substrate must only � DMG Them. Cell lysates were also analyzed for levels and total SHIP1 actin. PtdInsP2 ELISA bone marrow neutrophils from wild-type and SHIP1 � � �� � ice were resuspended in PBS �m container