Il 1β ab9722

Equal amounts of EVs pellets (10 μg), cell lysates (15 μg) and tissue protein (20 μg), as determined by BCA protein estimation, were boiled with 5× Loading buffer and run on a 12% SDS-PAGE denaturing gel. The proteins were transferred to PVDF membranes. Blots were blocked with 5% BSA for 1 h at room temperature. Blots were probed with primary antibodies [HMGB1 (ab18256), RAGE (ab7764), NLRP3 (ab214185), ASC (ab47092), Caspase-1 (ab1872), IL-1 β (ab9722) (Abcam, Cambridge, UK), GSDMD (ab209845), Transferrin receptor (10084-2-AP, Proteintech, Rosemont, USA), β-actin (60008-1-Ig, Proteintech, Rosemont, USA), GAPDH (60004-1-Ig, Proteintech, Rosemont, USA), Histone H3.1 (17168-1-AP, Proteintech, Rosemont, USA)] diluted in blocking buffer overnight at 4 °C. Blots were washed 3 times with TBS-T and then incubated with HRP conjugated anti-rabbit or anti-mouse Antibody (Li-cor, Nebraska, USA) diluted in blocking buffer at room temperature for 1 h. Blots were washed thoroughly 3 times with TBS-T and was followed by detection with Odyssey and quantitated using a Molecular Dynamics Densitometer with LANE 1D software.

Kidney and cell lysates were extracted with extraction buffer or sample buffer as described previously^{43 (link)}. Protein samples were subjected to immunoblotting analysis with antibodies against α-SMA (100M4795; Sigma-Aldrich, St Louis, MO, USA), ASC (sc-22514-R; Santa Cruz Biotechnology, Dallas, TX, USA), caspase-1 (sc-514; Santa Cruz Biotechnology), F4/80 (MCA497GA; AbD Serotec, Raleigh, NC, USA), IL-1β (ab9722; Abcam, Cambridge, MA, USA), Nrf2 (sc-13032; Santa Cruz Biotechnology) and GAPDH (sc-25778; Santa Cruz Biotechnology). Signals were detected using an enhanced chemiluminescence system (GE Healthcare Japan, Tokyo, Japan). Each western blot was performed on a pooled sample of 4–6 mice from each group.

A TUNEL assay kit (#11684817910) was purchased from Roche (Mannheim, Germany). Streptozotocin (STZ) was purchased from Sigma. An ROS assay kit (#S0033) was obtained from Beyotime (Shanghai, China). NLRP3 (ab214185), NeuN (ab104224) and IL-1β (ab9722) antibodies were purchased from Abcam (Cambridge, UK). ASC (D2W8U) and TXNIP (D5F3E) antibodies were obtained from Cell Signaling Technology (MA, USA). Caspase 1 (AF5418) and cleaved caspase-1 (AF4005) antibodies were purchased from Affinity.β-Actin and GAPDH antibodies were purchased from Bioss (Beijing, China). Genipin (Sigma-Aldrich, MO, USA) was diluted to appropriate concentrations in cell culture medium. ELISA kits were purchased from Abcam.