Ephrin a5

The cell immunofluorescence staining was performed following the procedure reported by another research group [37 ]. Primary antibodies against ADAM10 (CST, Boston, USA), EphA3 (Santa Cruz, California, USA), ephrin-A5 (R&D systems, Minnesota, USA) were applied at a ratio of 1:200. Fluorescent secondary antibodies (Alexa488, Alexa594, Thermo Scientific, Massachusetts, USA) were also applied at a ratio of 1:200. The images were observed and collected using a laser confocal microscope.

DIV3 neurons were treated with Sema 3 A (250 ng/ml, Peprotech, # 150–17 H) or Ephrin A5 (1 µg/ml, R&D Systems, # 374-EA) for 1, 5, 15, or 30 min in neuronal growth media, fixed for 15 min with 4 % PFA/4 % sucrose, and washed in PBS. Neurons were permeabilized with 0.2 % Triton X-100 in PBS for 10 min and blocked in antibody buffer (4 % BSA, 0.1 % Tween-20 in PBS) for 60 min at room temperature. Neurons were stained with primary antibodies overnight at 4 °C and with a mix of secondary antibodies and phalloidin for two hours at room temperature. Neurons transfected with plasmids expressing Halo-tagged AnkB440 proteins were treated with fresh media containing Sema 3 A and with the JF 549 HaloTag ligand (1:200) for 30 min. The percent of collapsed growth cones was recorded for each experimental condition, selecting for transfection when applicable. Axon growth cones were recorded as collapsed based on the presence of a pencil-like shape devoid of lamellae and with a maximum of one filopodia, or intact based on the fan-shaped morphology enriched in filopodia and/or lamellipodia.

Cells were divided into the positive control group, negative control group and experimental group; and the protein lysate of PCa cells was extracted according to the literature [38 (link)], 1.5 μg antibody [ADAM10 (CST, USA), EphA3 (Santa Cruz, USA), ephrin-A5 (R&D systems, USA), or ephrin-A5 (Santa Cruz, USA) was added to the test group and 1.5 μg homologous lg monoclonal antibody was added to the negative control group. After incubated in a shaker at 4 °C for 12 h, the target protein was enriched by 20 μl washed protein A/G agarose magnetic beads(MCE, USA). The precipitated proteins were detected using Western blotting.

Ephrin-A5 was purchased from R&D and dissolved in water at 1 mg ml⁻¹ stock concentration and further diluted at a final concentration of 500 ng ml⁻¹ in the recording medium. Forskolin (Sigma Aldrich) was dissolved in DMSO at 10 mM concentration and further diluted in the recording medium at 25 μM. IBMX (Sigma Aldrich) was dissolved in DMSO to a 100 mM concentration and further diluted at 100 μM in the recording medium.
HEK293 cells (from ATCC, not authenticated, not tested for mycoplasma contamination) were used to validate the plasma membrane localization of Lyn- and Kras-targeted constructs. Although this cell line is commonly misidentified, this did not affect the conclusion drawn using it since similar experiments were reproduced using retinal neurons.