Anti human il 1β

The sample buffer was added to the cell lysates and boiled at 100°C for 10 minutes. The same amount of protein was loaded into SDS−PAGE gel wells. The gel was electrophoresed at 80 V for 0.5 hours and 120 V for 1 hour. Proteins were transferred from gels onto PVDF membranes (Millipore, Massachusetts, USA) at 90 V for 1 hour. The membranes were blocked with 5% nonfat milk at room temperature for 2 hours and then incubated overnight with the following primary antibodies in primary antibody dilution buffer at 4°C: anti-IL-1β (R&D Systems Cat# AF-401-NA, RRID: AB_416684), anti-caspase-1 (AdipoGen Cat# AG-20B-0042, RRID: AB_2490248), anti-NLRP3 (AdipoGen Cat# AG-20B-0014, RRID: AB_2490202), anti-ASC (Cell Signaling Technology Cat# 67824, RRID: AB_2799736), anti-human IL-1β (Cell Signaling Technology Cat# 83186, RRID: AB_2800010), anti-human caspase-1 (Cell Signaling Technology Cat# 3866, RRID: AB_2069051), and anti-β-actin (ZSGB-Bio Cat# TA-09, RRID: AB_2636897). The membrane was washed three times with PBST for 5 minutes each and then incubated for 1 hour at room temperature using conjugated secondary antibodies (1:5000) in blocking buffer. The membrane was washed three times with PBST for 5 minutes each time. Images were obtained after chemiluminescence visualization.