Rabbit anti asc

The L4-L5 segments of the spinal cord were homogenized and subjected to SDS-PAGE as previously described48 (link). The membranes were incubated with the following primary antibodies at 4 °C overnight: goat anti-IL-1β (1:500; R&D), rabbit anti-caspase-1 (1:500; Abcam), rabbit anti-NALP1 (1:500; Abcam), rabbit anti-ASC (1:500; sc-22514-R; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-mIL-1β (1:500; sc-23460; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-pSTAT3 (1:500; Cell Signaling Technology), rabbit anti-STAT3 (1:1000; 79D7; Cell Signaling Technology, USA), rabbit anti-pJAK2 (1:500; 3771; Cell Signaling Technology, USA), rabbit anti-JAK2 (1:1000; 3230; Cell Signaling Technology, USA), and mouse anti-β-actin (1:5000; 4967; Cell Signaling Technology, USA). The blots were then washed in TBST and incubated in the appropriate secondary antibody for 1 hour at room temperature. The secondary antibodies were donkey anti-goat lgG-HRP (1:10000; sc-2020; Santa Cruz Biotechnology, CA, USA), donkey anti-mouse lgG-HRP (1:5000; sc-2314; Santa Cruz Biotechnology, CA, USA), or HRP Affinipure Goat Anti-Rabbit lgG (H+L) (1:10000; E030120-01; ErthOx, CA, USA). The western blot images were captured on an ImageQuant LAS4000 mini image analyser (GE Healthcare, Buckinghamshire, UK), and the band levels were quantified using the Image J software, version 1.42q.

This test was performed as we described in our previous paper [50 (link)]. Hippocampus lysates (500 μg) were immunoprecipitated with 1 μg of anti-ASC antibody (Santa Cruz) overnight at 4 °C, then incubated with 30 μl of protein A agarose beads (Cell Signaling) at 4 °C for 4 h, and centrifuged at 12,000×g for 60 s. Protein complexes were washed five times with RIPA buffer, resuspended in 2× loading buffer, and heated at 95 °C for 10 min before western blot analysis by using the following antibodies: rabbit anti-ASC (1:1000, Santa Cruz), rabbit anti-NALP1 (1:200: Abcam), rabbit anti-NLRP3 (1:200, Santa Cruz), and rabbit anti-caspase-1 (1:200: Santa Cruz). Whole tissue lysate prepared for IP (50 μg) was used as an input, and homophytic IgG as the negative control.

GES-1 cells that adhered to the round glass coverslips were fixed in 4% buffered paraformaldehyde and permeabilised with 0.1% Triton X-100. The cells were then incubated using goat anti-NLRP3 (1:200, Abcam), mouse anti-CASPASE-1 (1:200, Santa Cruz) and rabbit anti-ASC (1:100, Santa Cruz). After incubation with primary antibodies, the samples were washed and labeled with corresponding Alexa Fluor-488- and Alexa Fluor-555-conjugated secondary antibodies. The cells were visualized under a Zeiss LSM800 microscope. Colocalization was analyzed by Image Pro Plus software. Colocalization coefficient was represented by Pearson’s correlation coefficient as described.