Anti nlrp3 cryo 2

Cell pellets were lysed in Laemmli buffer ×2 (Tris–HCl 0.5 M, pH 6.8; 0.5 M DTE; 0.5% SDS) and protein concentrations determined using the Bradford reagent (Bio-Rad). Protein extracts were separated on SDS–PAGE (8% or 15% or 4–15% gradient [vol/vol]) gels. Gels were transferred onto nitrocellulose membranes (GE HealthCare and Bio-Rad) for immunoblotting with the following antibodies: anti-NLRP3 (Cryo-2, 1:1,000) and anti-caspase-1 (Bally-1, 1:1,000) from AdipoGen, anti-ASC (1:2,000) from Enzo Life Science, and anti-γH2AX (JBW301, 1:1,000), anti-P-Ser15-p53 (1:1,000), and anti-ATM Ser1981 (10H11.E12, 1:1,000) from Millipore. Anti-P-KAP1 Ser824 (1:1,000), anti-KAP1 (1:1,000), and anti-NEK7 (A302-684A, 1:1,000) from Bethyl Laboratories, anti-p53 (clone DO7 1:2,000) and anti-NOXA (114C307, 1:1,000) from Santa Cruz; anti-ATM (#ab32420, 1:1,000), anti-fibrillarin (ab4566, 1:1,000), and anti-GAPDH (ab9484, 1:1,000) from Abcam; anti-FLAG (F7425 1:5,000) and anti-α-tubulin (clone B-5-1-2 1:1,000) from Sigma-Aldrich; anti-XPO2 (GTX102005 1:1,000) and anti-IPO5 (GTX114515 1:1,000) from Genetex, and anti-actin (C4, 1:100,000) from MP Biomedical. The Fiji and Image Laboratory (Bio-Rad) programs were used for densitometric analysis of immunoblots, and the quantified results were normalized as indicated in the figure legends.

Retinas were fixed in 4% paraformaldehyde (E672002-0500; Sangon Biotech, Shanghai, China) for 15 min at room temperature immediately after being resected from the mice, after which they were washed three times in PBS. Retinas were then flat-mounted, blocked, and permeabilized in blocking buffer for 1 h at room temperature. Isolectin GS-IB4 (I21411; Invitrogen, Carlsbad, CA, USA) was applied and retinas were left to incubate at 4°C overnight, followed by further incubation with anti-NLRP3 (cryo-2, AdipoGen) at 4°C overnight. The anti-mouse secondary antibody (ab150115, Abcam) was applied at room temperature for 1 h. After three washes with PBS, samples were mounted using the Vectashield HardSet mounting medium (H-1700-10; Vector Laboratories, Burlingame, CA, USA). The retinal vasculature was imaged using a confocal fluorescence microscope (TSC SP8; Leica Microsystems, Wetzlar, Germany). The density and lesions of the microvasculature were analyzed using the Image J software.