Tgx precast polyacrylamide gels

Unless specified otherwise, samples were heated in Laemmli buffer (from Bio-rad, no. 1610747) for 5 min at 95 °C, with or without 50 mM dithiothreitol (DTT; from GoldBio), and electrophoretically resolved under denaturing conditions on 4–20 % TGX precast polyacrylamide gels (from Bio-Rad). For Coomassie-blue staining, gels were first fixed in 50 % v/v methanol and 10 % v/v acetic acid for 30 min at RT, then incubated for a further 30 min in 50 % v/v methanol, 10 % v/v acetic acid, and 0.001 % w/v Coomassie Brilliant Blue R-250 (from BioRad), and finally destained overnight in 10 % v/v acetic acid before being washed into water. For immunoblotting, proteins were transferred overnight onto nitrocellulose membranes (from Bio-Rad, no. 1620112). Membranes were blotted in TBST buffer (20 mM Tris•NaOH pH 7.5, 150 mM NaCl, and 0.1 % v/v Tween 20) containing 3 % w/v non-fat milk solids (from Apex, no. 20241) using antibodies against the FLAG epitope (no. F7425) or the SBP tag (no. MAB10764), and fluorescently-labeled secondary antibodies IRDye 800CW or IRDye 680CW (from LI-COR Biosciences), as appropriate. Blots were imaged on a LI-COR Odyssey M imaging system.

Hippocampus and cortex was microdissected from frozen brains and processed to collect both soluble and insoluble extracts. Briefly, microdissected tissue was homogenized in TPER (ThermoFisher) and centrifuged at 12,000 RPM for 15 min. Supernatant was collected as the soluble fraction and the pellet was treated with 70 % formic acid and spun down at 25,000 rpm. The resulting supernatant was collected as the insoluble fraction. Insoluble protein samples were neutralized for Western blotting and further precipitated with trichloroacetic acid (TCA) when probing for insoluble tau. Protein samples were denatured at 95 °C for 15 min before being loaded onto 4-20 % TGX precast polyacrylamide gels (Bio-rad). Antibodies used for western blotting include: HT7 (1:1000), PS199 (Abcam; 1:1000), PS202 (Abcam; 1:1000), AT100 (ThermoFisher; 1:1000), AT270 (ThermoFisher; 1:1000), PHF1 (1:1000), 6E10 (1:1000), GFAP (1:1000). Mesoscale Discovery immunoassay kits (Mesoscale Diagnostics) were used for cytokine (K15048G) and Aβ38, 40, and 42 (K15199E) quantification of cortical samples following the manufacturers protocols. The proinflammatory MSD was able to detect levels of that were within the standard curve, whereas brain levels of IFN-γ, IL-12p70, CXCL1 and IL-4 were below the threshold of detection.