Rabbit anti asc

The BV2 cell lysates (500 μg) were immunoprecipitated with 1 μg of anti-ASC antibody (Cell Signaling Technology, 67824S) for 1 h at 4 °C, and then were incubated with 20 μl of protein A agarose beads (Santa-Cruz, sc-2003) overnight at 4 °C, and centrifuged at 3000×g for 5 min. Protein complexes were washed five times with RIPA buffer, resuspended in × 2 loading buffer, and heated at 95 °C for 5 min. Then, the protein lysis buffers were used for western blot analysis with the following antibodies: rabbit anti-ASC (Cell Signaling Technology, 67824S, 1:1000), rabbit anti-NLRP3 (Cell Signaling Technology, #8242S, 1:1000), and mouse anti-caspase-1 (Santa-Cruz Biotechnology, sc-398,715, 1:100). Homophytic IgG was used as the negative control. SDS-PAGE and Western blot were used to analysis IP assay. The ASC protein was used as a loading control, and the loading quantities of precipitated materials were regulated according to the gray levels of ASC protein, to ensure brightness of reference bands were consistent. As the molecular weight of pro-caspase-1 and ASC were close to 50 kD or 25 kD, to avoid the influence of IgG light or heavy chain, the second antibodies used for pro-caspase-1 and ASC were anti-Mouse IgG Light Chain (Abbkine, A25012) and anti-Rabbit IgG Heavy Chain (Abbkine, A25222), respectively.

Rat mesangial cells (HBZY-1) were obtained from the Wuhan cell repository (Wuhan, China). Naringin (purity of ≥98%) was obtained from Cayman (CA, USA), stored at 2–4 °C, and protected from light. MCC950 was purchased from Selleckchem (CA, USA). IL-1β and IL-18 ELISA kits were purchased from R&D Systems (USA). Rabbit anti-NLRP3 and rabbit anti-ASC were obtained from Cell Signaling Technology (CST; CA, USA). Rabbit anti-caspase-1 and GAPDH were obtained from Santa Cruz (CA, USA).

Nigericin (CAS number 28643-80-3) and Caspase-1 inhibitor VX-765 (Cat# inh-vx765i-5) were purchased from the InvivoGen Biotech Co., Ltd. (USA). The monoclonal antibodies mouse anti-FLAG (Cat#8146), mouse anti-Myc (Cat#2276), mouse anti-β-actin (Cat#3700), mouse anti-HA (Cat#2367), rabbit anti-IL-1β (Cat#12703), rabbit anti-NLRP3 (Cat#15101), rabbit anti-Casp-1 (Cat#24232), and rabbit anti-ASC (Cat#67824) were purchased from Cell Signaling Technology (USA). Rabbit polyclonal SVA 3D antibody was prepared by us using purified SVA 3D protein. MCC950 (Cat# HY-12815A) (NLRP3 activation inhibitor) was purchased from MCE (Shanghai, China). The translation inhibitor CHX (Cat# A8244) was purchased from APEXBIO (Houston, TX, USA). Lipofectamine 2000 (Cat# 11668019) was purchased from the Thermo Fisher Scientific, USA.