Neural growth factor

The BioID assay was performed based on the description of Roux et al.^{74 (link)}. The CAR-BirA* construct was stable transfected into PC12 cells using zeocin as selection antibiotic. Details on the DNA constructs and the assay is available online. In brief, stable BioID clones were differentiated for 3 days with 50 ng/ml neural growth factor (Sigma-Aldrich) prior to incubation with 50 µM biotin for 3 days. Biotinlylated proteins were enriched using Streptavidin-Dynabeads and identified by Massspectrometry.

Sensory neuron axon growth of DRGs was evaluated by assessing the length of outgrown axons. Lumbar DRGs were dissected and cultivated for 3 days in a neurobasal medium infused with B27 (2%, Gibco), fetal horse serum (2%, PAN-Biotech), glutamine (1%, Thermo Scientific), gentamycin (0.5%, Gibco) and neural growth factor (10 ng/ml, Sigma-Aldrich) on poly-d-lysine (0.2 mg/ml, Sigma-Aldrich) and laminin (1 µg/ml, Sigma-Aldrich)–coated slides in a 5% CO₂-humified atmosphere. Axonal growth was determined after 72 h by fixation in 4% PFA and stained with primary antibody beta-III-tubulin (1:2000, Sigma-Aldrich, RRID: AB_262133). Imaging was performed with an inverted fluorescence microscope (BX51, Olympus). Axon lengths were measured with the NeuronJ plugin for ImageJ. Three to four DRGs of each rat were recorded and analysed.