Crispr cas9 protein

Amine-terminated and ethylenediamine-cored G5 PAMAM (theoretical molecular mass: 28,826 Da) was purchased from Dendritech (Midland, MI). Benzoic acid, dicyclohexylcarbodiimide, N-hydroxysuccinimide, rhodamine B isothiocyanate (RBITC), Cyt C from equine heart, saporin, and lysozyme from chicken egg white were purchased from Sigma-Aldrich (St. Louis, MO). 4-(Bromomethyl)phenylboronic acid was purchased from Meryer Chem. (Shanghai, China). β-Gal and RNase A were purchased from J&K Scientific (Shanghai, China). FITC, trimethylamine, trypsin from porcine pancreas, and Amplex Red were purchased from Macklin (Shanghai, China). BSA and hydrogen peroxide were purchased from Aladdin (Shanghai, China). CRISPR-Cas9 protein was purchased from GenScript (Nanjing, China). T7E1 enzyme was obtained from New England Biolabs (Beijing, China). PULSin was purchased from Polyplus-transfection (France). TransEx was obtained from Cenji Biotech (Shanghai, China). Lipofectamine CMAX Transfection Reagent was bought from Thermo Fisher Scientific (Germany). R-phycoerythrin (R-PE) was purchased from Cayman Chemical (Michigan, USA). GFP, YFP, and RFP were gifts from N. Chen in CAS. HRP and TMB were purchased from Yuanye Biotechnology (Shanghai, China). LysoTracker Red (DND-99, Invitrogen) was purchased from Thermo Fisher Scientific (Germany).

DNCB was obtained from Sigma-Aldrich (St. Louis, MO). Imiquimod cream [5 weight % (wt %)] was purchased from 3M Pharmaceuticals (Aldara; Leicestershire, UK). Dex cream was purchased from CR Sanjiu (Shenzhen, China). Tacrolimus ointment was purchased from Astellas (Toyama, Japan). Penicillin-streptomycin, PBS, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (USA). HA was obtained from Bloomage Freda Biopharm Co. Ltd. (Shandong, China). CTP was supplied by Jellice Co. Ltd. (Miyagi, Japan). CRISPR-Cas9 protein was purchased from GenScript (Nanjing, China). T7E1 enzyme was obtained from New England Biolabs (Beijing, China). Lipofectamine CMAX Transfection Reagent was bought from Thermo Fisher Scientific (Germany). sgNLRP3 was designed by online tools (http://crispr.mit.edu/ and http://chopchop.cbu.uib.no/) and then prepared using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, MA, USA). Primers for in vitro transcription of gRNAs are listed in table S1. Primary antibodies used in this project included the following: Anti-NLRP3 (1:1000; ER1706-72) antibody was obtained from HUABIO (Hangzhou, China), anti–caspase-1 p10 (1:1000; sc-514) and anti–glyceraldehyde-3-phosphate dehydrogenase antibodies were obtained from Santa Cruz Biotechnology (CA, USA), and anti–IL-1β (1:1000; ab200478) was obtained from Abcam (Shanghai, China).