Bs6067

Heart tissues or HL-1 cardiomyocytes were homogenized in RIPA lysis buffer and centrifuged at 12,000g at 4 °C for 25 min. Then, protein supernatants were obtained and added with loading buffer to boil for 10 min. A total of 10 to 30 µg proteins were used to perform western blot as previously reported^{33 (link)}. Primary antibodies for NLRP3 (CST, 15101), pro-caspase-1 (CST, 24232), cleaved-caspase-1 (CST, 89332), GSDMD (Abcam, ab219800), cleaved N-terminal GSDMD (CST, 10137), IL-1β (Bioworld, BS6067), IL-18 (Bioworld, BS6823) were used, respectively. β-tubulin was used as an internal reference for protein analysis.

To measure the levels of interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), VEGF-C, VEGFR3, and LYVE-1, equal amounts of the samples were loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels. Then, the resolved proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and incubated for one hour, at room temperature, in blocking solution (5% nonfat dried milk dissolved in Tris-buffered saline with Tween 20 [TBST] buffer). Then, the filters were probed, overnight, at 4°C, in blocking solution containing primary antibodies, diluted 1 : 1,000, against the following: IL-6 (MB9296, Bioworld), IL-1β (BS6067, Bioworld), TNF-α (11948S, Cell Signaling Technology), VEGF-C (sc-374628, Santa Cruz Biotechnology), VEGFR3 (ab27278, Abcam), LYVE-1 (ab14917, Abcam), and β-actin (AP0060, Bioworld). Membranes were washed twice with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology), for 1 hour, at room temperature, followed by washing three times. Signal detection was performed using an enhanced chemiluminescence substrate (Millipore, USA) and quantitated using Image J software.