Col18a1

RPPA were obtained as described previously [46 (link)]. Samples were lysed using Tissue Protein Extraction Reagent (Thermo Scientific, Waltham, MA, USA) and diluted to 0.5 mg/mL with Novex Tris-Glycine SDS Sample Buffer 2X (Invitrogen Corporation, Carlsbad, CA, USA Thermo Scientific). Each lysate was spotted in 4-point two-fold dilution curves onto nitrocellulose-coated microscope slides (Grace Biolabs, Bend, OR, USA) using an Aushon Arrayer 2470 (Aushon Biosystems, Billerica, MA, USA). Slides were incubated with a single pre-validated primary antibody using DAKO Autostainer Plus (DAKO Corporation, Glostrup, Denmark). They underwent signal amplification (CSA kit, DAKO) and staining with streptavidin-conjugated IRDye680LT® (LI-COR Biosciences, Lincoln, NE, USA), before scanning on a Tecan Power Scanner (Männedorf, Switzerland) equipped with a customized emission filter to improve the IRDye680LT® fluorescence collection efficiency. Image analysis for spot recognition, quantification and normalization was carried out using MicroVigene 5.2 software (Vigene Tech Inc., Carlisle, MA, USA). For RPPA analysis, antibodies for COL18A1, GRFα2, ITIH4, PCOLCE were from Sigma-Aldrich Ltd (The Old Brickyard, New Road, Gillingham, Dorset SP8 4XT) and antibodies for HRG, MGP, NPDC1 and ribonuclease A were from Abcam 330 (Cambridge Science Park, Cambridge CB4 0FL, UK).

For each CSF sample, protein concentrations were measured with the bicinchoninic acid (BCA) assay (Bio-Rad Laboratories Srl, MI, IT). The protein content of the samples ranged from 0.25 μg/μl to 1.25 μg/μl, and 20 μg of each sample were loaded on 4-12% NuPAGE Bis-Tris gradient mini gels (Life Technologies, Grand Island, NY).
Protein electroblotting was performed following standard procedures, using antibodies for IGSF8, ITIH4, PCOLCE, FGA, FGG, GFRalpha2 and COL18A1 from Sigma-Aldrich Ltd (The Old Brickyard, New Road, Gillingham, Dorset SP8 4XT); for COL1A2, HRG, IGFBP4, MGP, NPDC1 and ribonuclease A from Abcam 330 (Cambridge Science Park, Cambridge CB4 0FL, UK); for selenoprotein P from Santa Cruz (10410 Finnell Street Dallas, Texas 75220 USA). WB bands were quantified with Quantity One software (Bio-Rad, USA) in terms of density (total intensity/area of volume).