6 m urea

The washed beads were resuspended in 500 μL 6 M urea (Sigma) in PBS and incubated with 10 mM DTT (GoldBio) for 20 min at 65 °C. Each sample was then treated with 20 mM iodoacetamide (IA; Sigma) for 30 min at 37 °C, diluted with 950 μL PBS, and pelleted by centrifugation (1,400 rcf, 3 min, RT). The supernatant was discarded, and the beads were incubated with 200 μL of a pre-mixed solution containing 2 M urea in PBS, 1 mM CaCl₂, and 2 μg Sequencing Grade Trypsin (Promega). The beads were shaken overnight at 37 °C and pelleted by centrifugation (1,400 rcf, 3 min, RT). The beads were then washed sequentially with PBS (500 μL x 3) and ddH₂O (500 μL x 3), resuspended in sodium dithionite (50 μL of a 50 mM stock solution in ddH₂O; Sigma), and incubated at RT for 1 h. The beads were pelleted by centrifugation (1,400 rcf, 3 min, RT), and the supernatant was transferred to a new tube. The remaining beads were twice incubated with sodium dithionite (75 μL of 50 mM in ddH₂O) and pelleted as before, and the resulting supernatants were transferred to the same tube. The beads were then washed twice with PBS (100 μL), and each wash was combined with the previous supernatants to give a total volume of ~350 μL. The cleaved peptides were treated with 1/20 volume of formic acid (Sigma), and the samples were stored at −20 °C until LC/LC–MS/MS analysis.

We assessed Total and p-Tau using the western blot technique at 12DIV and 24DIV. Hippocampal slices (n = 96; 48 females) were evenly distributed across control and ethanol groups at 12DIV and 24DIV. In order to amplify the signal, slices from membranes within respective conditions and sex were grouped and scraped from the membrane into chilled 1X PBS buffer containing: protease inhibitor cocktail III (1:200) (MilliporeSigma) and 10-mM sodium fluoride (1:100) (Sigma). Tissue was then lysed in lysis buffer containing: 62.5-mM tris base (Fisher Scientific; Hampton, New Hampshire, United State), 6-M urea (Sigma), 10% glycerol (Fisher Scientific), 2% SDS (Bio-Rad; Hercules, California), protease inhibitor cocktail III (1:200) (MilliporeSigma), and 10-mM sodium fluoride (1:100) (Sigma). Samples were placed in ice for 30 min. Following dissociation, samples were centrifuged at 13,000 × g for 10 min at 4 °C. Supernatant was separated and stored at −80 °C for later use.