Il 1β sc 12742

Angiotensin II (10 μM, A9525) and sauchinone (SML0783) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Four 488 phalloidin, dichlorofluorescin diacetate (DCFH-DA), DAPI, primary antibodies for p-Smad-2 (44-244G), p-Smad3 (44-246G), and NLRP3 (MA5-23919) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies for CDK2 (SC-6248), CDK4 (SC-23896), cyclinD1 (SC-8396), cyclinE (SC-247), p21 (SC-397), p27 (SC-1641), Smad-4 (SC-7966), CTGF (SC-373936), ICAM-1 (SC-8439), NfκB p65 (SC-8008), p-IκB-α (SC-8404), ASC (SC-514414), and IL-1β (SC-12742) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MCP-1 (44-246G) was purchased from Abcam (Cambridge, MA, USA). Capase-1 (4199) and HRP conjugated secondary antibodies raised against mouse, rabbit, and goat were purchased from Cell Signaling Technology (Danvers, MA, USA).

Protein extracts were prepared from mouse brain or primary cortical neurons as previously described [4 (link), 5 (link)]. Protein extracts from brain tissue (50 µg) and primary neurons (30 µg) were subjected to SDS-PAGE gels, electroblotted onto Immobilon P membrane (Millipore, Bedford, MA, USA), and incubated with specific antibodies against S100β (sc-136061, Santa Cruz Biotech.), receptor for advanced glycation end-products (RAGE, sc-365154, Santa Cruz Biotech.), glyoxalase 1 (sc-133214, Santa Cruz Biotech.), interleukin-1β (IL-1β, sc-12742, Santa Cruz Biotech.), tumour necrosis factor-α (TNF-α, 500-P64, Peprotech) or sLeX (CD15s, 551,344, BD Pharmingen), followed by incubation with the specific horseradish peroxidase–conjugated anti-mouse or anti-rabbit (Bio-Rad Lab.) secondary antibodies. The immunocomplexes were visualized by chemiluminescent detection in a ChemiDoc™ MP imaging system from Bio-Rad Laboratories. Staining with anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sc-32233, Santa Cruz Biotech.) or β-actin (A5541, Sigma) was used as a loading control. The relative intensities of protein bands were analyzed using the Image Lab™ analysis software from Bio-Rad Laboratories.

Cell culture materials were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against p-STAT3 (Tyr705) (#9145, 1:1000), STAT3 (#30835, 1:2000), p-p65(Ser536) (#3033, 1:1000), p65 (#8242, 1:2000), and α-SMA (#19245, 1:2000) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against IL-1β (sc-12742, 1:2000), CD34 (sc-7324, 1:2000), VCAM-1 (sc-13160, 1:2000), and EPAS-1 (sc-13596, 1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against CD68 (28058-1-AP), ICAM-1 (60299-1-Ig, 1:2000), MCP-1 (66272-1-Ig, 1:2000), Bax (50599-2-Ig, 1:2000), Bcl-2 (12789-1-AP, 1:2000), cleaved-caspase-3 (19677-1-AP, 1:2000), GAPDH (10494-1-AP, 1:2000), FN1 (15613-1-AP, 1:2000), and SOX9 (67439-1-Ig, 1:2000) as well as secondary antibodies (Goat anti-mouse, SA00001-1; Goat anti-rabbit, SA00001-2) were purchased from Proteintech Group (Chicago, IL, USA). Recombinant human interleukin-1 receptor antagonist (IL-1Ra) (SRP3084) was obtained from Sigma (St. Louis, MO, USA), Stattic (inhibitor of STAT3) and Colivelin, (activator of STAT3) were purchased from MedChemExpress (Shanghai, China). Unless otherwise indicated, the remaining reagents used in this study were obtained from Sigma.