Iblot2 apparatus

Total proteins were extracted using RIPA buffer (Euromedex, Souffelweyersheim, France), and then equal amounts of proteins were reduced, size-separated on 12% stain-free precast SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA), and transferred to nitrocellulose membranes by using an iBlot2 apparatus (Thermo Fisher Scientific). The membranes were blocked in 5% milk in TBS-Tween 0.1% and incubated with specific primary antibodies overnight at 4°C; the antibodies were against ACE2 (AF933, R&D Systems, Minneapolis, MN, USA; 1:200), phospho- and total p38 (9211 and 9212, Cell Signaling Technology, Danvers, MA, USA; 1:2,000), phospho- and total NF-κB p65 (3039 and 8242, Cell Signaling Technology; 1:2,000), and β-actin (A2228, Sigma-Aldrich; 1:5,000). The blots were exposed to horseradish peroxidase-conjugated anti-rabbit (Cell Signaling Technology, 7074; 1:10,000) and anti-goat (A27104, Thermo Fisher Scientific; 1:2,000) secondary antibodies, and bound antibodies were detected using Clarity chemiluminescent substrate (Bio-Rad). Images were recorded using a Fujifilm LAS-3000 bioimaging system (Stamford, CT, USA).

Total cell lysates (50 μg) were resolved on Bolt 8% Bis–Tris Plus gels, which were electrophoretically transferred to a PDVF membrane with the iBlot2 apparatus (Thermo Fisher Scientific, Carlsbad, CA). Following 1 hour in blocking buffer [5% dry milk, TTBS (Tween Tris buffered saline, TBS + 0.05% Tween)], blots were stained with Cell Signaling (Danvers, MA) antibodies against HER2/c-erb-B2 (D8F12) and b-actin (13E5), followed by a goat anti-rabbit secondary antibody (7074). All blots were washed 3 times with TTBS after antibody staining. Bands were detected with the Amersham ECL Prime chemiluminescent system (Cytiva, Marlborough, MA) and visualized with film.

The urea fractions were diluted in LDS sample buffer (Thermo Fisher) supplemented with reducing agents (Thermo Fisher) and boiled for 5 min. Proteins were separated on 10% NuPAGE pre‐cast Bis‐Tris gels (Thermo Fisher), transferred using an iBlot2 apparatus (Thermo Fisher). Membranes were Ponceau‐stained, then blocked using protein free Blocking buffer (Thermo Fisher), and probed overnight at 4°C with a polyclonal TDP‐43 C‐terminus antibody recognizing TDP‐43 at epitopes from amino acid 260 onwards (Proteintech, 12892‐1‐AP) or phosphorylated TDP‐43 at S409/410 (Proteintech, 22309‐1‐AP) and 2 h at room temperature with secondary antibody rabbit HRP. Antibody signal was detected with ECL reagent (GE Healthcare) using a ChemiDoc MP Imaging System (Bio‐Rad). For interpretation of the immunoblots the ratios of the intensities of the CTFs over full‐length TDP‐43 were compared.

Proteins were extracted from snap-frozen tissues and cell pellets, and western blotting (WB) was performed using Bolt 4–12% Bis-Tris gels (cat# NW04125BOX, ThermoFisher Scientific), an iBlot 2 apparatus (cat# IB21001, ThermoFisher Scientific) and an iBind apparatus (cat# SLF1000 or SLF2000, ThermoFisher Scientific), as described [27 (link)]. Primary antibodies used are outlined in Table 2. Secondary antibodies included: HRP-linked goat anti-rabbit (1:1000; cat# ab6721, Abcam), HRP-linked goat anti-rabbit (1:1000; cat# 111-035-045, Jackson Immunology), and Alexa Fluor^® 488 donkey anti-mouse (1:1000; cat# A-21202, ThermoFisher Scientific).
Membranes were imaged using a ChemiDoc MP Imaging System (Biorad) and ImageLab 6.0 software (Biorad). Densitometry was performed using ImageLab 6.0, with the intensity values for the protein-of-interest normalized against α-tubulin. Densitometry data were analyzed using GraphPad Prism 8 (San Diego, CA, USA).

Proteins were separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane using iBlot 2 apparatus (Thermo Fisher Scientific). The membrane was incubated in TBST (20 mM Tris, 150 mM NaCl, and 0.1% Tween 20) containing 5% milk for 1 h, washed three times with TBST, and incubated with the primary antibody—the polyclonal anti-Dam1p (Keating et al., 2009 (link)) or monoclonal anti-GFP antibody (Sigma; 11814460001) overnight at 4°C. The blots were incubated with either HRP-tagged secondary antibody (Sigma; A3415-.5ML; Cell Signaling Technologies; 7076P2) or IRDye secondary antibody (LI-COR, 926–68074 and 926–32212). The blots were then scanned using either with ChemiDoc (Bio-Rad) or LI-COR odyssey imager.

When required, lysed samples were subjected to quantitative Western analysis. Unless stated otherwise, generally samples were prepared by mixing 80 μl resuspended cells with 30 μl 4× LDS buffer (Thermo Fisher Scientific) and 10 μl 1 M dithiothreitol. The samples were then incubated at 70°C for 10 min. A small volume (10–15 μl) of lysed samples was loaded onto precast 4–12% Bis-Tris NuPAGE gels (Thermo Fisher Scientific) and run at 200 V for 60 min using MES or MOPS running buffer (Thermo Fisher Scientific). Gels were then washed with deionized water and blotted onto nitrocellulose membranes using the iBlot2 apparatus (Thermo Fisher Scientific) according to the manufacturer’s instructions. The membranes were then incubated for 60 min in block solution (2.5% skimmed milk powder in TBS buffer, pH 7.5) before overnight incubation at 4°C with primary antibodies diluted in block solution supplemented with 0.1% Tween-20. Membranes were then washed three times for 5 min in TBS-T (0.1% Tween-20 in TBS) before incubation with secondary antibodies conjugated to AF647 or CF770 far-red/infrared dyes (1:1,000) for 60 min at room temperature. The membranes were subsequently washed three times for 5 min with TBS-T, once for 5 min with TBS, and then fluorescently imaged using the LI-COR Odyssey or Azure c600 imaging systems, according to the manufacturer’s instructions.