Furthermore, under physiological conditions, with all limitations of our methods, we did not detect alterations in neuronal maturation and synaptic protein composition

Furthermore, under physiological conditions, with all limitations of our methods, we did not detect alterations in neuronal maturation and synaptic protein composition. Supplementary Physique 3: Analysis of the subplate in Ns-/- mice at E16. (A) Nissl staining of the developing cortex of wild-type and Ns-/- mice at E16. (B) The same region was stained with an antibody directed against the neuronal marker MAP2 (green). Nuclei were counterstained with DAPI (blue). CP, cortical plate; SP, subplate. Scale bar: 50 m. Image_3.TIF (1.0M) GUID:?9C053C8C-AF27-4943-8F79-B2ECAC0D6C56 Supplementary Figure 4: Quantification of Reelin, ADAMTS-4, and PAI-1 expression and cleavage. Intensity of each SR1001 band was normalized to -actin expression. Relative expression is usually presented (AU, arbitrary models), expression for the wt group was set to 1 1. = 3 animals; three technical replicates were analyzed; bars indicate mean, error bars SR1001 indicate SD. Image_4.TIF (499K) GUID:?06EC09AC-96C6-4D3C-8A5F-43D8A358493F Supplementary Table 1: Animals used in this study. Number and age of the mice SR1001 included in this study are listed, as well as brain area and analysis that has been performed with the tissues. Table_1.XLSX (11K) GUID:?C07B625A-5998-4B48-9F6F-CFAE1562C78C Supplementary Table 2: List of proteins identified by differential quantitative proteomics. Synaptosomes were isolated from the neocortex of adult neuroserpin-deficient mice and control littermates and analyzed by differential quantitative proteomics to assess synaptic alterations between both mouse groups (= 4). In red are proteins with the PRIDE (https://www.ebi.ac.uk/pride/) partner repository with the dataset identifier PXD022371. Prior to mass spectrometric analyses, peptides were resuspended in 0.1% FA to a final concentration of 1 1 g/l. LC-MS/MS measurements were performed on a quadrupole-iontrap-orbitrap mass spectrometer (Orbitrap Fusion, Thermo Fisher) coupled to a nano-UPLC (Dionex Ultimate 3000 UPLC system, Thermo Fisher). One micro gram of tryptic peptides were injected into the chromatographic system an autosampler, purified and desalted using a reversed phase trapping column (Acclaim PepMap 100 C18 trap; 100 m 2 cm, 100 ? pore size, 5 m particle size; Thermo Fisher) and transferred to a reversed phase column for chromatographic separation (Acclaim PepMap 100 C18; 75 m 50 cm, 100 ? pore size, 2 m particle size, Thermo Fisher). Trapping was done for 5 min at a flow rate of 15 l/min with 99% solvent A (0.1% FA) and 1% solvent B (0.1% FA in ACN). Separation and elution of peptides were achieved by a linear gradient from 1 to 30% solvent B in 70 min at a flow rate of 3 l/min. Eluted peptides were ionized using a nano-electrospray ionization source (nano-ESI) with a spray voltage of 1 1,800, transferred into the mass spectrometer and analyzed in data dependent acquisition (DDA) mode. For each MS1 scan, ions were accumulated for a maximum of 120 ms or until BCL2L a charge density of 2 105 ions (AGC Target) was reached. Fourier-transformation-based mass analysis of the data from the orbitrap mass analyser was performed covering a mass range of 400C1,300 m/z with a resolution of 120,000 at m/z = 200. Peptides with charge says between 2+ and 5+ above an intensity threshold of 1 1,000 were isolated within a 1.6 m/z isolation windows in Top velocity mode for 3 s from each precursor scan and fragmented with a normalized collision energy of 30% using higher energy collisional dissociation (HCD). MS2 scanning was performed using an orbitrap mass analyser, covering a mass range of 380C1,500 m/z with a orbitrap resolution of 15’000 at m/z = 200 and accumulated for 60 ms or to an AGC target of 1 1 105. Dynamic exclusion of fragmented peptides was applied for 15 s after precursor selection. Natural data obtained from LC-MS/MS measurements were processed with MaxQuant version 1.6.2.10 (Max Plank Institute.