Fig 1: Quantitative proteome and phosphoproteome of the rat supraoptic nucleus.A, graphical representation of the experimental approach (1). Twelve adult Sprague Dawley rats were divided into two groups: six control with constant access to water and six subjected to a 48-h water deprivation protocol (WD) to activate magnocellular neurones (MCNs) (2). The supraoptic nucleus (SON, mainly containing MCN cell bodies and dendrites) was punched from the hypothalamus, and the neurointermediate lobe (NIL, mainly containing axonal terminals from the posterior pituitary as well as the intermediate lobe) was dissected from the anterior pituitary (3). Proteins from SON and NIL were extracted and processed for Nano-LC Mass Spectrometry (LC-MS/MS) (4). Proteomics and phosphoproteomic determinations were performed by LC-MS/MS. Generated using BioRender (https://biorender.com/). B, principal component analysis (PCA) of the SON proteome and phosphoproteome in control (blue, n = 6) and WD rats (red; n = 6). C, volcano plot of WD versus control SON proteome showing 247 upregulated (p-value <0.05, red) and 78 downregulated (p-value <0.05, blue) proteins. D, volcano plot of WD versus control SON phosphoproteome showing 252 hyperphosphorylation (p-value <0.05, red) and 36 hypophosphorylation (p-value <0.05, blue) events. E, global overall phosphorylation state change (?Ps) analysis of phosphoproteins between control and WD rats in the SON. Numbers of hyperphosphorylated (Hyper) and hypophosphorylated (Hypo) peptides are shown. Dotted lines, ?Ps = ±0.34. F, Venn diagram showing 23 proteins in common with changes at the proteome and phosphoproteome level in response to WD. G, phospho raw abundance for S244-p S6, S1412-p NOS1, S25-p STMN1, and S255-p JUND in the SON according to LC-MS/MS between control (n = 6) and WD (n = 6) rats. H, immunohistochemistry against S244-p S6 in the SON of control and (H') WD rats. I, immunohistochemistry against S6 in the SON of control and (I') WD rats. J, immunohistochemistry against S1412-p NOS1 in the SON of control and (J') WD rats. K, immunohistochemistry against NOS1 in the SON of control and (K') WD rats. L, immunohistochemistry against S25-p STMN1 in the SON of control and (L') WD rats. M, immunohistochemistry against STMN1 in the SON of control and (M') WD rats. N, immunohistochemistry against S255-p JUND in the SON of control and (N') WD rats. Images are representative of n = 4. Scale bar represents 25 µm. LC-MS/MS, Nano-LC mass spectrometry.