Western Blot Analysis of Inflammasome Proteins

Twelve microliters of supernatant or cell lysate were separated on a 4–12% Mini-Protean TGX gel (Bio-Rad) at 100 V for 1 h. Proteins were transferred onto a 0.2 μm nitrocellulose membranes (Li-Cor Biotechnology) at 100 V for 1 h. The membranes were blocked in 5% non-fat dry milk in PBS containing 0.2% Tween-20 for 1 h followed by incubation in PBS containing 5% BSA, 0.2% Tween-20, and either 1:1,000 rabbit polyclonal anti-Caspase-1 p20 (47 (link)), anti-IL-1β (70 (link)), anti-HMGB1 (abcam; ab18256), anti-PKR (phospho T446) (abcam; ab32036), anti-PKR (abcam; ab226819), anti-beta Actin antibody (abcam; ab8229), Anti-GFP antibody (abcam; ab6556), or 1:500 anti-Asc (Tyr-144) phospho-specific antibody (ECM Bioscience; AP5631) with rotation overnight at 4°C. The following day, donkey anti-rabbit IRDye 800CW and donkey anti-goat IRDye 680RD secondary antibodies (Li-Cor Biotechnology) were applied at a dilution of 1:15,000 with rocking for 1 h at room temperature. Membranes were imaged on a Li-Cor Odyssey CLx machine with auto exposure and high-quality setting.

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Darweesh M., Kamel W., Gavrilin M.A., Akusjärvi G, & Svensson C. (2019). Adenovirus VA RNAI Blocks ASC Oligomerization and Inhibits NLRP3 Inflammasome Activation. Frontiers in Immunology, 10, 2791.

Publication 2019

Mini-Protean TGX gel 0.2 μm nitrocellulose membranes ab18256 ab32036 ab226819 ab8229 ab6556 IRDye 800CW IRDye 680RD secondary antibodies Odyssey CLx machine

Anti antibodies Anti antibody Antibody Beta actin Caspase Cell Dilution Donkey Goat Hmgb1 Il 1β Irdye 800cw Membranes Milk Nitrocellulose Proteins Rabbit Tween 20

Corresponding Organization : Uppsala University

Other organizations : Al-Azhar University, The Ohio State University

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Variable analysis

independent variables

Amount of supernatant or cell lysate separated on the gel
Antibody used for immunoblotting (anti-Caspase-1 p20, anti-IL-1β, anti-HMGB1, anti-PKR (phospho T446), anti-PKR, anti-beta Actin, Anti-GFP, or anti-Asc (Tyr-144) phospho-specific)

dependent variables

Protein expression or phosphorylation levels detected by immunoblotting

control variables

Gel type (4–12% Mini-Protean TGX gel)
Voltage and duration of gel electrophoresis (100 V for 1 h)
Voltage and duration of protein transfer (100 V for 1 h)
Blocking solution (5% non-fat dry milk in PBS with 0.2% Tween-20)
Incubation buffer (PBS with 5% BSA and 0.2% Tween-20)
Incubation temperature and duration (overnight at 4°C)
Secondary antibody dilution (1:15,000)
Secondary antibody incubation time and temperature (1 h at room temperature)
Imaging system (Li-Cor Odyssey CLx machine with auto exposure and high-quality setting)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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