GDNF, NRTN, and ARTN induce SHEP cell adherence and SFK activation in an HSPG-dependent manner. (A) RT-PCR of the cDNA from SHEP cells with syndecan-3–specific primers. The resulting 140-bp band represents a syndecan-3 mRNA-derived PCR product (lane 2). (lane 1) Molecular weight markers with sizes in bp. (B) Crystal violet staining of SHEP cells plated on immobilized GDNF, ARTN, NRTN, and PSPN with or without heparinase III (H’aseIII) pretreatment. (C) Quantification of attached SHEP cells on immobilized GFLs. Some cells were treated with heparinase III. Quantification also includes controls with cell attachment on BSA and attachment of cells treated with heparinase III on tissue culture plates. (D) Quantification of SHEP cell adherence to immobilized GDNF and ΔN-GDNF. Cells were plated on GDNF untreated, or they were pretreated with PI-PLC, chondroitinase ABC (Ch’ase ABC), NCAM function-blocking antibodies (NCAM BLK), or GDNF function-blocking antibodies (GDNF BLK). (C and D) Error bars show SEM from three independent assays. (E) Western blot (WB) for activated SFK in lysates from SHEP cells plated on GDNF, ΔN-GDNF, or BSA. In control experiments, cells plated on GDNF were treated with heparinase III (HIII) or PI-PLC. Western blots were probed with anti-pY⁴¹⁸Src (top) or anti-Src (bottom) antibodies. (F) SHEP cell adherence and spreading on immobilized GDNF in the absence or presence of 2 µM SFK inhibitor SU6656. Cell spreading on immobilized GDNF was impaired in the presence of the inhibitor, whereas adherence of SHEP cells was not significantly affected by SU6656. (G) Adherence and spreading of SHEP cells infected with adenovirus expressing GFP (AdGFP) or dominant-negative Src (AdDN-Src/GFP) on immobilized GDNF. Transduction of cells with AdDN-Src/GFP resulted in SHEP’s failure to spread on immobilized GDNF. GFP-expressing adenovirus did not affect SHEP cell spreading on the GDNF matrix. Untreated, UNTR. Bars, 100 µM.