Supersignal west pico plus chemiluminescent substrate

Cells were lysed in a lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton-X 100 and a Halt^TM Protease Inhibitor Cocktail (100x, Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured by a Qubit^TM Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Equal amount of proteins was run on 10% Tris-tricine gel and blotted to polyvinylidene fluoride (PVDF) membrane with a Bio-Rad Wet Blotting System (Bio-Rad Hungary, Budapest, Hungary). The HER2 receptor was detected by an ErbB2 (HER2) antibody cocktail (Thermo Fisher Scientific, MA5-14057, produced in mouse, 1:500) and an anti-mouse-horseradish peroxidase (HRP) secondary antibody (Thermo Fisher Scientific, 32430, produced in goat, 1:500). As a loading control, β-actin was detected by an anti-actin antibody (Santa Cruz Biotechnology, Dallas, TA, USA, sc-1616, produced in goat, 1:2500) and an anti-goat-HRP secondary antibody (Santa Cruz Biotechnology, sc-2354, produced in mouse, 1:2000). After the addition of enhanced chemiluminescence (ECL) substrate (SuperSignal^TM West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific), the chemiluminescent signal was detected by ChemiDoc XRS+ Detection System (Bio-Rad Hungary).

Nitrocellulose membranes (Merck Millipore, Schaffhausen, Switzerland) were plotted with 3 μL of 1 mg/mL of either rIAPP, hIAPP monomer, hIAPP fibrils, or a control amyloidogenic protein amyloid beta (Aβ; Bachem, Bubendorf, Switzerland). After drying, the membrane was boiled at 900 W for 5 min in a 4.3 mM Na₂HPO₄ 1.4 mM KH₂PO₄ containing a buffer (pH 7.2) to expose epitopes and increase antigen binding; it was then washed and blocked with 50 mM Tris-buffer containing 0.1% Tween20 and 2.5% casein. Overnight incubation with the purified m81 mAb was performed. Finally, a secondary anti-mouse IgG directly conjugated with Horse-Radish Peroxidase (Jackson ImmunoResearch, Cambridgeshire, UK) was used. Development of the membrane was performed using a SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Basel, Switzerland), and pictures were acquired with an Azure C300 Imaging System (Axon Lab, Baden, AG, Switzerland). Fiji Image J was used to quantify the intensity of the dots. A background subtraction of 25 pixel was applied before quantification of the intensity or density. Monomeric IAPP and IAPP/Aβ fibrils were assessed with Thioflavin, as described above.

Nitrocellulose membranes (Millipore Corporation, US) were plotted with 3 μL of 1 mg/mL of either rIAPP, hIAPP monomer, hIAPP fibrils or a control amyloidogenic protein amyloid beta (Aβ; Bachem, Switzerland). After drying, the membrane was boiled at 900 W for 5 min in a 4.3 mM Na₂HPO₄ 1.4mM KH₂PO₄ containing buffer to expose epitopes and increase antigen binding; washed and blocked with 50 mM Tris-buffer containing 0.1% Tween 20 and 2.5% casein. Overnight incubation with the purified polyclonal antibody was performed and finally a secondary anti-mouse IgG directly conjugated with horseradish peroxidase (Jackson Immuno Research Laboratories, Baltimore Pike, West Grove, PA, USA) was used. Development of the membrane was performed using a SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Federal Way, WA, USA) and pictures were acquired with an Azure C300 Imaging System (Axon Lab AG, Baden, Switzerland). Fiji Image J was used to quantify the intensity of the dots. A background subtraction of 25 pixel was applied before quantification of the intensity od density. Monomeric IAPP and IAPP/Aβ fibrils were assessed with Thioflavin as described above.

Expression of mitochondrial fusion OPA1, MFN1, MFN2 and fission DRP1, and FIS1 proteins was measured by western blot. HAECs were seeded in 6-well plates, treated as above, and lysed on ice in RIPA buffer supplemented with protease and phosphatase inhibitors cocktail. Total protein was quantified by bicinchoninic acid. Obtained protein was electrophoresed and transferred to PVDF membranes. Membranes were incubated overnight at 4 °C with the antibodies and dilutions mentioned in Table 1. The next day, membranes were washed and incubated with anti-rabbit IgG HRP-linked or anti-mouse IgG HRP-linked (Table 1) antibodies at room temperature for 1 h. Protein bands were detected with SuperSignal^TM west pico PLUS chemiluminescent substrate and captured by the iBright 1500 imaging system from ThermoFisher Scientific (Chelmsford, MA, USA). Analysis of obtained bands was evaluated by densitometry with Image J software [38 (link)].

Western blotting (WB) was carried out routinely. The primary antibodies used here included anti-Oct4 (1:1000, #2750; Cell Signaling), CD144 (1:1000, #2500; Cell Signaling), CD105 (1:1000, ab11414; Abcam), CD31 (1:1000, #3528; Cell Signaling), mouse anti-human GAPDH (1:10000, HRP-60004; proteintech) and mouse anti-human β-actin monoclonal antibody (1:10000, A5316; Sigma). Protein bands were visualized with SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (#34580; Thermo).

Conformational antibody characterization was performed by dot blotting using BclxLnf37, BclxLnf70, BclxLcf37, Bcl-xL-ΔTM monomer, Aβ fibers and Aβ monomer as a target on nitrocellulose membranes (Hybond-ECL—Amersham Biosciences). Following a short centrifugation, 10 µg of BclxLnf70, BclxLnf37, BclxLcf37, or Aβ fibrils and of Bcl-xL-ΔTM monomer were spotted on the membrane and dried for 30 min. After blockage at room temperature with PBS-Tween 20/10% milk and washing in PBS-Tween 20/5% milk, the membranes were incubated with either a rabbit polyclonal anti-Bcl-xL Ab (BD Bioscience; 1/2000) or mouse monoclonal anti-Bcl-xL Ab _(3–14) (obtained against the N-terminal part (3–14) of Bcl-xL; BD Bioscience; 1/2000) or with the mAbs to be characterized (1/5000) in PBS-Tween 20/5% milk. After three washes, they were incubated with a secondary goat anti-rabbit or rabbit anti-mouse Ab coupled to HRP (horseradish peroxidase) (BETHYL laboratories; 1/5000) in PBS-Tween 20/5% milk. Binding was revealed with a mixture of the SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (ThermoScientific #34579). Signals were observed using the Molecular Imager device (Fusion FX7, ThermoFischer).

Lysates from mouse tissues were made in TNE buffer supplemented with protease and phosphatase inhibitors. After sonication, a clarifying spin (5000 × g for 10 min at 4 °C), and protein quantification with a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific; Waltham, MA), 20 µg of total cell lysate was separated by SDS-PAGE on an 8% polyacrylamide gel using a Bio-Rad gel electrophoresis system following the manufacturer’s protocol. Separated proteins were transferred onto PVDF membranes using the Bio-Rad Trans-Blot Turbo Transfer System. Western blotting analysis was performed using an mCNT1 (Alomone Labs; ANT-061, 1:500) primary antibody with GAPDH (CST 97166, 1:5000) as a loading control. Rabbit (Bethyl; A120-201P, 1:5000) and mouse (Bethyl; A90-116P, 1:5000) secondary antibodies were used, and proteins were visualized using the SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific; Waltham, MA) on a ChemiDoc MP Imaging System from Bio-Rad. Uncropped blots are provided in the Data Source file.