Phl1a pcdna3

The following plasmids were obtained from Addgene (Cambridge, MA, USA): pUltra-Chili (48687) and pUltra-Chili-Luc (48688, gifts from Malcolm Moore), pSpCas9(BB)-2A-GFP (48138, gift from Feng Zhang [47 (link)]), pcDNA3-S33Y β-catenin (19286, gift from Eric Fearon [48 (link)]), and phL1A-pcDNA3 (12307, gift from Vance Lemmon [49 (link)]). M50 Super 8x TOPFlash (12456) and M51 Super 8x FOPFlash (12457, gifts from Randall Moon [50 (link)]) contain, respectively, eight copies of consensus or mutant TCF/LEF DNA binding sites upstream from sequences encoding firefly luciferase [50 (link)]. The Renilla luciferase-encoding plasmid pRL-CMV (E2261) was purchased from Promega (Madison, WI, USA). The small hairpin RNA (shRNA) transfer vectors containing control or FER-targeting shRNA sequences (FER[978-998]) were generated as described [20 (link),51 (link)]. Lentiviruses encoding GFP and dox-inducible control or FER-targeting shRNAs were packaged in COS-7 cells, as described [52 (link)]. To generate lentivirus encoding firefly luciferase and tdTomato fluorescent protein, COS-7 were transfected with pUltra-Chili-Luc and lentivirus packaging plasmids, as described [52 (link)].

COS7 cells were seeded at a density of 5 × 10³ cells/cm² on collagen IV-coated cover glass 24 h before transfection. Total 1.3 µg of DNA was mixed for each 1 mL culture medium. Nrp1 (pCherry-mNrp1, #21934, addgene), L1CAM (phL1A-pcDNA3, #12307, addgene), and SLITRK1 or its mutant (pEF-SLITRK1-ires-alkaline phosphatase or its derivatives) plasmids were mixed at a ratio of 2:3:3. Transfection was done using polyethyleneimine (Polysciences, #23966)^{70 (link)}. Five hours after transfection, the medium was changed to fresh DMEM (10% FBS, 1% antibiotic–antimycotic [15240062, Gibco]). Twenty-four hours after transfection, 10 µg/mL of recombinant Sema3a-Fc (5926-S3, R&D) and 5 µg/mL of FM4-64 dye (F34653, Thermo Fisher) were added to the culture medium and incubated for 30 min at 37 °C in 5% CO₂ incubator. Cells were washed with a conditioned medium and with cold PBS(−) sequentially and were fixed with ice cold PFA (4% PFA, 100 mM NaCl, 100 mM sodium phosphate, pH 7.0) for 30 min on ice. Cells were washed with PBS(−) and incubated in blocking buffer (2% normal goat serum, 3% BSA, 0.1% Triton X-100 in PBS[-]). L1CAM was detected with anti L1CAM (MAB5272, Chemicon, 1:1000) and Alexa488-conjugated anti-rat IgG. Sema3a-Fc was detected with Alexa633-conjugated anti-mouse IgG. Cell images were obtained using a confocal microscope (Zeiss LSM-800) and analyzed by ImageJ.