Low‐temperature SDS‐PAGE

Dimerization of iNOS was separated by using low‐temperature SDS‐PAGE. Rat insulin‐producing INS1E cells were seeded at a density of 3 × 10⁶ cells per well in six‐well plates. After pretreatment with different concentrations of CAT639 for 4 h, cells were treated with 10 ng·mL⁻¹ IL‐1β for 24 h. Then cells were lysed on ice with cell lysis buffer (Cell Signaling, Danvers, MA) and sonicated for 5 s. The supernatant was cleared by centrifugation at 4°C and 16 000× g. Protein samples were prepared with LDS non‐reducing sample buffer without heating. SDS‐PAGE was run on ice at constant 125 mA for 5 h with pre‐cooled NuPAGE SDS running buffer. Proteins were transferred by wet transfer at constant 70 mA for 2.5 h. The following Western blot steps were done as usual.