Cells with neurites shorter than the diameter of its soma were excluded from the analysis

nscription was verified via PCR reaction using specific primers for GAP-DH. Finally the corresponding cDNAs of one sample were combined and used as one template. Precipitation of extracellular proteins from COS-7 cells and subcellular membrane fractionation The culture medium of COS-7 cells, expressing Syndecan-2int.myc-GFP, was completely removed and after washing with pre-warmed HBSS replaced with 5 ml HBSS+ containing 20 mg/ml Brefeldin A. After 30 min, the cells were supplemented with 20 mM Y-P30 or with the appropriate volume of 5 mM Tris-HCl, pH 7.4 as a control. In order to inhibit matrix metalloproteinase activity either 50 nM GM6001 or 20 nM of the specific MMP9/13Inhibitor I were added. Three hours later the HBSS-medium was harvested and centrifuged at 30006g. 5 ml of the remaining supernatant were mixed with 20 ml freezing ethanol and incubated at 220uC over night. On the next day, the precipitated proteins were centrifuged at 4uC and 10.000 rpm for 10 min and the resulting pellet was washed twice with 15 ml 80% ethanol. Residual ethanol was removed by lyophilizing the pellets for 5 min. Afterwards the protein pellet was dissolved for 3 h at 4uC in 100 ml ultrapure water containing 26EDTA-free complete proteinase inhibitor and finally solubilized by adding 100 ml 26SDS sample buffer and boiling for 5 min at 95uC. For subcellular membrane fractionation corresponding pellets from COS-7 cells were re-suspended in 800 ml HOM-buffer and subsequently homogenized with 15 strokes at 900 rpm in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19653943 a Potter S homogenizer. The resulting homogenates were centrifuged in 1.5 ml Eppendorf tubes for 10 min at 10006g at 4uC. Afterwards, the supernatants were transferred in fresh tubes and kept on ice. The pellets were re-suspended again and the procedure repeated. In the next both supernatants of the respective sample were combined and spun for 30 min at 20.0006g. The resulting pellets were again re-suspended in 800 ml HOMbuffer, homogenized with 12 strokes at 900 rpm and centrifuged for 30 min at 20.0006g. The obtained pellets represent a crude membrane fraction and were re-suspended in 800 ml 5 mM Tris, pH 8.1, containing 0.32 M sucrose and PI and were subsequently loaded on top of a 1.1 M/1.4 M sucrose step gradient and finally centrifuged for 2 h at 85.0006g and 4uC in a ultra centrifuge. Subsequently, the membrane-containing fraction was transferred into a fresh tube and the sucrose concentration adjusted to 0.32 M Quantitative Real-Time PCR Quantitative Real-Time PCR was performed using the LightCycler 1.5 Instrument from Roche. For the reaction mix the LightCycler TaqMan Master kit was used according to the manufacturers instructions. qRT-PCR reactions were started with an initial denaturation step for 10 min at 95uC, followed by 45 cycles of 95uC for 10 sec, 60uC for 60 sec and 72uC for 1 sec. Relative amounts of Reelin and GluN2B had been normalized with Hprt for mRNA amount variations. The mRNA expression is presented as the change of relative quantities and was analyzed using the 2-DDCt method. Expression- and knock down constructs Full-length cDNAs of SDC-2 and SDC-3 were cloned into the pEGFP-N3 Vector or pcDNA3.1 Myc-HisA and expressed in COS-7 cells. The successful MedChemExpress A-83-01 incorporation in cellular membranes was verified microscopically and via subcellular fractionation. In order to detect the extracellular domains separately, additionally a mycepitope tag was cloned into the Hind III restriction site of SDC-2 and Pst I restriction site o