Criterion vertical electrophoresis cell

Protein was isolated from cells using RIPA Buffer (Sigma Cat# R0278) or sorted directly into Laemmli Buffer (BioRad Cat# 1610747). Western blot analysis was performed using the Criterion Vertical Electrophoresis Cell (BioRad Cat# 1656020) with 4–15% Criterion Tris-HCl Protein Gels (BioRad Cat# 3450028) and imaged with the Azure Biosystems c300. Western blots were quantified by ImageJ densitometry analysis.

The separation of proteins was performed by two-dimensional electrophoresis (2DE) according to the two-dimensional electrophoresis step-by-step user instructions (BioRad, Hercules, CA, United States). The specific steps are as follows: 700 μg of total protein from each sample was diluted to up to 300 μL with Hydration loading buffer I (containing Dithiothreitol (DTT) and Bio-Lyte). Each mixture was loaded onto a 17 cm precast immobilized pH gradient (IPG) gel strip (pH gradient 4-7). The first-dimension separation-isoelectric focusing (IEF)-was then carried out in the Protean IEF Cell (Bio-Rad, Hercules, CA, United States). IPG DryStrips were equilibrated in a reducing agent followed by an alkylating agent. The second dimension was performed by placing the strips on 12% acrylamide gels (Bio-Rad, Hercules, CA, United States) to allow protein separation by electrophoresis in a Criterion™ Vertical Electrophoresis Cell (Bio-Rad, Hercules, CA, United States). The analytical gels were visualized with Bio-Rad Laboratories GS-710 Calibrated Imaging Densitometer Scanner after Coomassie Brilliant Blue G-250 staining. The digitalized 2-DE gel images were studied (protein spot detection, spot matching, and semi-quantitative statistical analysis) using PDQuest 2-D Analysis Software (Bio-Rad, Hercules, CA, United States).

Cardiomyocytes were lysed and proteins quantified using Pierce™ BCA Protein Quantification Kits. Proteins (30 µg) were loaded onto Bio-Rad Criterion™ 10% pre-cast mini gels and separated by electrophoresis using XT MOPS running buffer and a Bio-Rad Criterion™ Vertical Electrophoresis Cell. Proteins were blotted onto a nitrocellulose membrane using a Criterion™ Blotter (1 h at 100 V at 4°C). Membranes were blocked for 1 h at RT before being divided and probed with anti-GPER (1:500), anti-ERα (1:5,000), anti-ERβ (1:1,000), and for housekeeping anti-GAPDH (1:20,000, sc-47724, Santa Cruz Biotechnology) overnight at 4°C. Next, membranes were washed with Tris-buffered saline with 0.1% Tween^® 20 before the application of secondary antibody (1:2000, anti-rabbit IgG-HRP, 7074S, Cell Signalling Technology or anti-mouse IgG-HRP, ab205719, Abcam) for 1 h at RT. The membranes were washed and visualised using Bio-Rad Clarity Western ECL Substrate and captured using the Bio-Rad ChemiDoc MP.

Tau-441 (2N4R) (rPeptide, T-1001-1; molecular weight, 45.9 kDa) (2 μg) was reconstituted in 1% NH₄OH and digested by 100, 10, 1, and 0.1 nM Kgp/RgpB in working buffer [20 nM sodium phosphate and 1 mM DTT (pH 7.5)]. Digestion reactions were performed for 1 hour at 37°C; reactions were stopped with protease inhibitor (P8340, Sigma-Aldrich). Proteins were separated by electrophoresis at 70 V for 15 min and then at 85 V for 1.5 hours on a 10% Criterion TGX precast gel (Bio-Rad, 5671033) (Bio-Rad, Criterion vertical electrophoresis cell) and electroblotted overnight onto a PVDF membrane at 10 V (Bio-Rad, Criterion Blotter). Blot was blocked with BLOTTO (87530, Thermo Fisher Scientific) for 1 hour and probed with primary antibody anti-Tau46 (13-6400, Thermo Fisher Scientific) at 1:1000 dilution in 3% BSA in TBS for 2 hours. Blot was then washed three times for 10 min each with TBST (28630, Thermo Fisher Scientific) and then incubated with secondary antibody HRP goat anti-mouse (1:50,000; 31439, Thermo Fisher Scientific) in 3% BSA in TBS for 30 min. After further washing, blot was washed three times for 10 min with TBST, and blot staining was visualized using chemiluminescence detection (34096; SuperSignal West Femto, Thermo Fisher Scientific).

Small pieces of gray matter, 70–100 mg in total (wet mass), were dissolved in 2× Laemmli Sample Buffer and applied on 4–15% precast polyacrylamide gel (Criterion TGX), together with prestained Protein ladder EXtended PS13 (5–245 kDa, GeneON, Ludwigshafen, Germany) and positive control samples (cultured primary neurons from adult goat spines treated with the apoptosis-inducing agent staurosporine). Separation was performed in Criterion™ Vertical Electrophoresis Cell (BioRad, Hercules, CA, USA) with a constant voltage 140 V. Proteins from the gel were transferred onto Immobilon-FL PVDF membranes (0.45 µm, Millipore now Merck KGaA, Darmstadt, Germany) blocked with Pierce Protein-Free Blocking Buffer (Thermo Scientific, Waltham, MA, USA), cut on the level of 75 kD standards lines and stained separately with primary antibodies against active caspase 3 (ab214430, AbCam, Cambridge, UK) and neurofilaments (anti-Neurofilament M antibody, Merck/Chemicon, Darmstadt, Germany) as loading control. They were then developed with Amersham ECL Prime Reagent and visualized from contact-exposed X-ray film. Only areas of interest were manually scanned (with GT-X980 scanner, Epson, Suwa, Nagano, Japan) from the films due to constraints of file size and optical density processing.