Mab casper 1

Cells were lysed in RIPA buffer (R&D Systems) containing protease inhibitors (Sigma-Aldrich) and total protein was determined by BCA (Thermo Fisher Scientific). Lysates or supernatants were separated by SDS-PAGE (NuPAGE, Thermo Fisher Scientific) and blotted onto nitrocellulose, nytrane membranes (GE healthcare, Chicago, IL, USA). Anti-mouse caspase-1, full-length and activated p20 fragment (mAb Casper-1, Adipogen Life Sciences, Liestal, Switzerland), ASC (anti-Asc, pAb (AL177), Adipogen Life Sciences), NLRP3 (mAB Cryo-2, Adipogen Life Sciences), IL-1 ß (anti-mIL-1β R&D Systems) were used as primary and horseradish-peroxidase-conjugated anti-rabbit and anti-mouse IgG (both Cell Signaling Technology, Beverly, MA, USA) as secondary antibodies. Chemiluminescent substrate (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) was used for visualization.

BMDM supernatants, BMDM lysates, whole cortex brain lysates, and PBS soluble brain supernatants were prepared by adding NuPAGE LDS sample buffer (Invitrogen) and 10% β-mercaptoethanol and boiling for 10 min at 85 °C. Samples were run on NuPAGE 4-12% Bis–Tris precast gels (Invitrogen), transferred to nitrocellulose membranes, and blocked with 5% milk in PBS plus 0.05% Tween 20 (PBST) for 1 h at room temperature. Membranes were probed with anti-Caspase-1 (p20) (mouse), mAb (Casper-1) (Adipogen, Cat. No. AG-20B-0042) or anti-IL-1β (Abcam, Cat. No. ab234437) in 5% milk in PBST overnight at 4 °C. Membranes were subsequently washed and incubated with IR Dye® 800 CW goat anti-mouse antibody (LI-COR, Cat. No. 926-32210) for 2 h at room temperature, washed, and scanned with the Odyssey CLx (LI-COR) imaging system. For lysates, membranes were washed and stripped using Restore™ Western Blot Stripping Buffer (Thermo Fisher Scientific). Membranes were subsequently reprobed with β-actin antibody (Cell Signaling Technology, Cat. No. 4970), incubated with IR Dye® 800 CW goat anti-rabbit antibody (LI-COR, Cat. No. 926-32211) and imaged as described above. The densities of the caspase-1 bands were normalized to the β-actin loading control using Fiji/ImageJ software [57 (link), 58 (link)].