Protein a g plus agarose

Briefly, cell lysates were prepared using immunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM sodium fluoride, 1 mM Sodium Orthovanadate] supplemented with protease inhibitors (100 μg/ml phenylmethylsulfonyl fluoride (PMSF), Sigma-Aldrich; Complete Protease Inhibitor Cocktail, Roche, Indianapolis, IN, USA), then incubated with rabbit IgG together with 20 μl resuspended protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min. at 4°C. The supernatants were incubated with polyclonal anti-GFP (Abcam, Cambridge, MA, USA) antibody with shaking for 1 hr at 4°C, followed by overnight incubation with protein A/G PLUS-Agarose beads. The beads were then washed vigorously with immunoprecipitation assay buffer for four times. The eluted samples were then separated by SDS-PAGE, and subjected to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis, as well as immunoblotting to detect clathrin heavy chain.
Reciprocally, we also used primary antibody anti-clathrin heavy chain for immunoprecipitation. Samples were then separated by SDS-PAGE and immunoblotted with primary antibody against GFP (NeoMarkers, Fremont, CA, USA).

Ultrapure lipopolysaccharide (LPS) (E. coli 0111:B4, cat. no. tlrl-3pelps), standard LPS (E. coli 0111:B4, cat. no. tlrl-eblps), ATP (cat. no. tlrl-atpl), nigericin (cat. no. tlrl-nig), and MSU (cat. no. tlrl-msu) were purchased from InvivoGen, lipofectamine 3,000 transfection reagent (cat. no. L3000015) was purchased from Thermo Fisher, C646 (S7152) was bought from Selleck Chemicals, mouse immunoglobin IgG protein (cat. no. ab198772) was purchased from Abcam, Protein A/G PLUS-Agarose (cat. no. sc-2003) was obtained from Santa Cruz, cell lysis buffer (CLB) (cat. no. 9803) was purchased from Cell Signaling Technology, and mouse IL-1β (cat. no. 88–7013), tumor necrosis factor-α (TNF-α) (cat. no. 88-7324), interleukin-6 (IL-6) (cat. no. 88-701364), and human IL-1β (cat. no. BMS22) ELISA kits were purchased from Thermo Fisher.
Anti–IL-1β (1:1,000, AF-401-NA; RRID:AB_416,684) was purchased from RD System, anti-NLRP3 (1:1,000, Cryo-2) and ASC (1:1,000, AL177) were purchased from Adipogen, anti–caspase-1 (1:1,000, ab179515), and anti-NEK7 (1:5,000, ab133514) were purchased from Abcam; Anti–β-actin (1:10,000, BH10D10), anti-p65 antibody (1:1,000, 8242), anti–p-p65 antibody (1:1,000, 3033), and GAPDH antibody (1:2,000, 5,174) were purchased from Cell Signaling Technology, and DyLight 488–labeled secondary antibody (1:50, A120-100D2) was purchased from InvivoGen.