Phdh1

Neuro-2a cells were cultured on coverslips. After one PBS wash, cells were fixed in 3.7% paraformaldehyde for 10 min. Then, cells were permeabilized with 0.5% TritonX-100 for 5 min, followed by the addition of 100 nM Phalloidin to the cells. Phalloidin staining protocol was followed according to manufacturer’s instructions (Cat. #PHDH1, Cytoskeleton Inc). For VASP staining, cells were blocked in 5% normal goat serum (NGS) for 15 min at room temperature. VASP antibody (13472-1-AP, Proteintech) was prepared in 5% NGS (diluted in 1:200) and incubated overnight at 4 °C. The next day, cells were washed with PBS and incubated with the secondary antibody (diluted in 1:500, 5% NGS) at room temperature for 45 min. The coverslips were mounted on glass slides using DAPI Fluoromount-G (Cat. #0100-20, Southernbiotech) and imaged using a Visitech iSIM superresolution module and Hamamatsu Quest camera on a Nikon Eclipse Ti-E with a 100x 1.45NA objective. Images were deconvolved using Microvolution, and neurite length was calculated using NeuronJ plugin in Fiji, as previously described (41 (link)). NeuronJ traces the neurite by finding two points, one at the start and one at the end and calculating the optimal intensity path. Neurite lengths were quantified using Prism graphpad with three independent biological replicates.