Anti asc

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.

For confocal analysis of inflammasome molecule colocalization or aggregation, the intestine tissue slides were first fixed in 4% paraformaldehyde in phosphate-buffer saline (PFA/PBS) for 15 min. After being permeabilized with 0.1% Triton X-100/PBS and rinsed with PBS, the slides were incubated overnight at 4°C with anti-NLRP3 (1:200, Abcam, MA, United States) and anti-ASC (1:50, Enzo, PA, United States) or anti-caspase-1 (1:100, Abcam). After washing, these slides were incubated with primary antibodies and were then incubated with Alexa-488- or Alexa-555-labeled secondary antibodies for 1 h at room temperature. The slides were mounted and subjected to examinations using a confocal laser scanning microscope (Fluoview FV1000, Olympus, Japan) with photos being taken, and the colocalization of NLRP3 with ASC or caspase-1 was analyzed by the Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, United States). The summarized data of molecular colocalization efficiency were expressed as correlation coefficient as described previously (Boini et al., 2014 (link)), which indicated the formation of NLRP3 inflammasome in the intestine.