Ab9722

Cells were seeded on the circular cover glass in 24-well plates and were pre-incubated with 25 and 50 μg/mL SPJ for 12 h and followed with 1 μg/mL LPS for another 12 h. After ending the incubation, cells were washed twice with PBS, followed by fixation with 4% paraform and then perforation with 0.1% Triton X-100 for 20 min. After blocking with 1% BSA, cells were incubated with the following primary antibodies at 4 °C overnight: anti-IL-1β (Abcam, ab9722, 1:2000), anti-TNF-α (Santa Cruz, SC-5746, 1:1000), anti-ARG1 (Proteintech, 16001-1-AP, 1:1000), TGF-β (Abcam, ab92486, 1:1000). Following incubation with primary antibody, cells were incubated with FITC-labeled secondary antibodies (Jackson ImmunoResearch) for 1.5 h. After washing, cells were mounted with 40, 60-diamidino-2-phenylindole (DAPI) and Prolong Antifade Reagent (G1401, Servicebio, China). Staining patterns were examined using a scanning fluorescence microscope (Leica Microsystems).

Western blot was as described previously [34] (link). We used antibodies against NF-κB (1∶700, t-NF-KB p65 ab131485, p-NF-kB p65, ab28856, Abcam, UK), TXNIP (1∶700, ab86983), NLRP3 (1∶500, ab109314), ASC (1∶700, ab64808), caspase-1 (1∶500, ab1872), IL-1β (1∶1000, ab9722), collagen I (1∶1000, ab34710), collagen III (1∶500, ab7778), and β-actin (1∶1000, sc8432, Santa Cruz, USA). The protein bands were developed by the use of chemiluminescence (Millipore, USA), and quantified by densitometric analysis (Quantity One, Bio-Rad, USA).