Knock down of SDC-2 and -3 abolishes the Y-P30 induced CASK translocation in primary cortical neurons.

(a+b) shRNA knockdown was tested in co-transfected, SDC-2-myc or SDC-3-myc over-expressing Cos7 cells. The knockdown effects of the two most efficient shRNA sequences for each SDC (a) are shown in representative Western-blots (b). Note the efficient knockdown of the respective SDC. Syndecans are glycoproteins and the different bands detected by the myc antibody probably represent differentially glycosylated protein isoforms. (c) In order to establish a Lentivirus-based knockdown system, the appropriate shRNA cassettes of the pRS vectors, containing the U6-polymerase III-promoter, were subcloned into the PacI restriction site of the FUGW vector. (d, e) In mature primary cortical cultures (DIV18), expressing the knockdown construct against SDC-2, the Y-P30 induced CASK translocation is completely abolished. Similar effects after Y-P30 supplementation were obtained in young primary cortical cultures (DIV8) expressing the SDC-3 knockdown construct, indicating different cellular effects of both SDC (n = 4–6).