Ne per nuclear and cytoplasmic kit

Parental or KI HeLa cells were cultured in 6-well or 12-well plates and pretreated with DMSO (0.1%) or pomalidomide in culture medium for the times indicated in each figure. Then, the cells were stimulated with 20 ng/ml TNF-α for the indicated times and harvested using TrypLE (Gibco/Thermo Fisher Scientific). Nuclear and cytoplasmic fractions were extracted using the NE-PER Nuclear and Cytoplasmic Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. The protein fractions were separated by SDS-PAGE and analyzed by immunoblot using specific antibodies.

RAW 264.7 cells or BMDM were plated in 100 mm dishes (1 × 10⁶) in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were allowed to grow for 24 h and after incubation, cells were treated with LPS (50 ng/mL) with and without UroA (50 µM) and UAS03 (50 µM) for 6 h. After treatments, culture medium was removed and washed with PBS. Cells were scraped and pelleted down in PBS. Supernatant was discarded and pellet was used for isolation of nuclear and cytosolic protein using NE-PER Nuclear and Cytoplasmic kit (Thermo Scientific; Cat #78833). Later nuclear protein (2 µg) was used for EMSA using Non-Radioactive EMSA Kits with IR Fluo-Probes for Nuclear factor kappa B p65 (Viagene Biotech Inc Cat # IRTF282 60).