Protein Detection and Quantification in Cells

For protein detection, cells were harvested and lysed at 4 °C in RIPA buffer [50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 0.1% SDS, 1 mM EDTA] supplemented with protease and phosphatase inhibitors plus 1 mM DTT (Sigma Aldrich, Stockholm, Sweden) as described previously [18 (link)]. Thirty µg total protein were loaded into each well of a NuPAGE 4–12% Bis-Tris gradient gel (ThermoFisher, Stockholm, Sweden) and subjected to electrophoresis. The proteins were then transferred to a Hybond low-fluorescent 0.2 µm PVDF membrane (Amersham, Buckinghamshire, UK), blocked for 1 h in Odyssey^® Blocking Buffer (PBS) (LI-COR), and stained overnight at 4°C with antibodies against NLRP12 (Abcam, Stockholm, Sweden) and GAPDH (ThermoFisher, Stockholm, Sweden) as loading control. The membranes were then probed with the goat anti-rabbit IgG (H+L) HRP-conjugated antibody (ThermoFisher, Stockholm, Sweden) or the goat anti-mouse IRDye 680RD antibody (LI-COR Biotechnology, Lincoln, NE, USA) and proteins were detected using Clarity™ ECL substrates (BioRad, Hercules, CA, USA) and Super RX-N film (FujiFilm Nordic AB, Stockholm, Sweden), or the LI-COR Odyssey^® CLx scanner operating with Odyssey^® Image Studio software (LI-COR Biotechnology).