Anti nek7 antibody

After lysis of cells in an ice-cold lysis buffer (400 mM NaCl, 25 mM Tris–HCl, pH 7.4, 1 mM EDTA, and 1% Triton X-100) containing protease and phosphatase inhibitors, lysates were centrifuged at 12,000g for 10 min in microfuge tubes and supernatant was incubated with anti-NEK7 antibody (Abcam, 1:1,000) or control IgG in a binding buffer (200 mM NaCl, 25 mM Tris–HCl, pH 7.4, 1 mM EDTA) with constant rotation at 4°C for 1 h. After adding 50 μl of 50% of Protein G bead (Roche) to lysates and incubating with rotation at 4°C overnight, resins were washed with a binding buffer. After resuspending pellet in a sample buffer and heating at 100°C for 3 min, supernatant was collected by centrifugation at 12,000g for 30 s, followed by electrophoretic separation in a NUPAGE gradient gel. Immunoblot analysis was conducted by sequential incubation with anti-NEK7 or -NLRP3 antibody as the primary antibody and then with horseradish peroxidase-conjugated anti-rabbit IgG or -mouse IgG. Bands were visualized using an ECL kit.

NEK7 (Locus ID 59125) mouse shRNA (Cat#TL515241, OriGene Technologies) was used for NEK7 knock-down in primary cortical neurons, and pGFP-C-shLenti (Cat#TR30021V, OriGene Technologies) was used as an NC. NEK7 knock-down was performed following the manufacturer’s instructions. Knock-down efficiency was analyzed using a western blot (WB) analysis with anti-NEK7 antibody (Abcam, Cambridge, UK; ab133514).