Western blot analysis.

Immunoblot analyses were performed following protocols described in our previously published studies (27, 38). Briefly, cells or tissues were homogenized and lysed using modified RIPA buffer. Proteins were normalized using a Bradford assay before loading onto a SDS-acrylamide gel. Protein (10–40 μg) was loaded into each well of 10%–18% acrylamide gels, which were run at 110 V for 2–3 hours at 4°C. Following separation, the proteins were transferred onto nitrocellulose membranes at 27 V for 18 hours at 4°C. After transfer, the membranes were blocked with LI-COR blocking buffer for 45 minutes and incubated in primary and then secondary antibodies following the manufacturer’s protocol. The primary antibodies included anti-Kv1.3 (Alomone Labs, 1:1000) (Research Resource Identifier [RRID]: AB_2040151), anti-Kv1.3 (MilliporeSigma 1:1000) (RRID: AB_2265087), anti–p-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_331641), anti-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_330713), anti–p-Kv1.3 (MilliporeSigma, 1:1000, catalog SAB4504254), anti-PKCδ (Santa Cruz Biotechnology, 1:500) (RRID: AB_628145), anti-NLRP3 (AdipoGen, 1:1000) (RRID: AB_2490202), and anti–active MAPK (Promega, 1:2000). The secondary antibodies included IR-800–conjugated goat anti–mouse IgG (LI-COR, 1:20000) and IR-700–conjugated goat anti–rabbit IgG (LI-COR 1:20,000).