Finally, fibroblasts from Src knock out mice also show reduced spreading (Kaplan et al

Finally, fibroblasts from Src knock out mice also show reduced spreading (Kaplan et al., 1995). In seeming contrast to our results, Liu et al. antiCrat IgG were from Cappel (Organon Teknika Corp., Durham, NC). Peroxidase-conjugated donkey antiCrabbit IgG, FITC-conjugated donkey antiC mouse IgG, and FITC-conjugated donkey antiCrabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). The polyclonal anti-GFP antibody was from Laboratories (Palo Alto, CA). Cell Culture and Transfection Mouse L cell fibroblasts were cultured in DME (Laboratories). Wild-type and C215S mutant chick PTP1B cDNAs were amplified by PCR using primers that create restriction enzymes sites for XhoI at the 5 end and XbaI at the 3 end. The resulting fragments were cloned in frame into the XhoI and XbaI sites of pEGFP-C3. The fusion proteins contained the GFP at the NH2 terminus of PTP1B. After verifying all the constructs by sequencing, stable L cell lines were selected with G418. L cells transiently expressing the GFPCPTP1B construct NVP-AEW541 were prepared using Lipofectin, and the cells were processed for analysis after 24C48 h. Reverse Rabbit polyclonal to Acinus Transcription PCR Expression of transfected chick PTP1B was assessed by reverse transcription PCR (RT-PCR). In brief, total RNA was isolated from cultured cells using a Qiagen kit (Chatsworth, CA) and reverse transcribed with Superscript II and oligo dT primers (FACScan? (Bedford, MA). Background fluorescence was assessed in cells immunostained with normal rat IgG (20 g/ml) as primary antibody, followed by FITC-conjugated goat antiCrat IgG. 5,000 cells per sample were analyzed. Immunofluorescence Microscopy LP, LWT, and LMU cells were grown on acid-washed round coverslips (and and and and and and and and and and and and and and with in in and and and with with and and D) LMU cells exposed to LPA and stained for actin (C) or 1-integrin (D). Note the formation of actin stress fiber (C) and induction of focal contacts (D) in LMU cells exposed NVP-AEW541 to LPA. Tyrosine phosphorylation of FAK is shown in the immunoblot below. LP, LWT, and LMU cells were plated on fibronectin, lysed after 15 min under denaturing conditions, and immunoprecipitated with anti-FAK antibody. The immunoprecipitates were fractionated by SDS-PAGE and analyzed by immunoblotting NVP-AEW541 with the 4G10 monoclonal antiphosphotyrosine antibody. Note the difference in the phosphorylation of FAK when LMU cells were plated in the absence or presence of LPA. The bottom panel shows the same blot stripped and probed with anti-FAK antibody. Numbers at the left indicate the position of molecular mass markers in kilodaltons. Bar, 20 m. To determine whether there is an increase in phosphorylation of FAK on tyrosine residues as a response to LPA, nonionic detergent lysates of LMU cells were immunoprecipitated with anti-FAK antibody, and the precipitates were separated by SDS-PAGE and immunoblotted with antiphosphotyrosine antibody. 15 min after plating LMU cells on fibronectin, in the presence of LPA, the levels of phosphorylated tyrosine residues on FAK are similar to those in LP cells (Fig. ?(Fig.14).14). The effects of LPA in inducing spreading, focal contact and stress fiber formation, and FAK phosphorylation require integrin ligation since they are not observed in cells grown on polylysine (not shown). The rapid response of LMU cells to LPA treatment rules out the possibility that the machinery essential for phosphorylation of FAK and paxillin and for assembly of focal contacts and stress fibers is defective in LMU cells. Discussion In this paper, we present data that suggest NVP-AEW541 a regulatory role for PTP1B in integrin-mediated adhesion and spreading. L cells expressing a catalytically inactive form of PTP1B display reduced attachment and are unable to spread on fibronectin. They assume an elongated spindle shape after prolonged culture but fail to form the typical flattened phenotype. Consistent with this altered morphology, LMU cells also NVP-AEW541 show a dramatic decrease in focal adhesions and actin stress fibers. They are able, however, to assemble actin into long filaments parallel to the long axis of the cell and short filaments in the submembrane region of cells and lamellipodia. Additionally,.