Supplementary Material for: Modular Phosphoprotein Signatures Link Rac1 Inhibition to Neurite Morphogenesis in a Dose-Dependent Manner

Precise regulation of neurite initiation, elongation, and branching is critical for neuronal network formation. Rac1, a key regulator of cytoskeletal remodeling, influences neurite morphogenesis through protein phosphorylation-mediated signaling, but the global phosphorylation landscape that governs Rac1-mediated morphogenesis remains unknown. To address this knowledge gap, we performed phosphoproteomics profiling of primary rat cortical neurons treated with 3, 10, or 30 µM of a Rac1 inhibitor for 48 hours to evaluate phosphoprotein dynamics. Phosphorylation levels of 167 signaling proteins were quantified using a targeted phospho-antibody array, and correlated with neurite count, length, and branch point count. Correlation analysis identified morphology-specific phosphoproteins, such as Tau, CREB, CaMK2, and GAP43, whose phosphorylation levels were significantly associated with neurite morphology features at specific inhibitor concentration. These results define a correlation-based framework linking phosphoprotein signaling to neurite morphology and offer novel insights into neurodevelopmental processes, neuronal disorders, and developmental neurotoxicity.