In 10-cm dishes, 293T (HEK 293T) cells (American Type Culture Collection) were cultured according to the manufacturer’s instructions until confluent. Cells were pretreated for 15 min with 1% hydroxypropyl-β-cyclodextrin (HPCD) in DMEM containing 1% delipidated serum. HPCD was removed, and cells were treated with 30 μM cSN50.1 or cSN50.1α in DMEM containing 5% delipidated serum for 2 h (see (9 (link)) for details) Nuclear extracts were obtained as described above. HPCD-stimulated cells not treated with peptide and unstimulated cells served as positive and negative controls, respectively.
The nuclear content of NF-κB RelA in RAW cells or SREBP2 in HEK 293T cells was determined by quantitative immunoblotting using rabbit polyclonal anti-NF-κB RelA (Abcam) or rabbit polyclonal anti–SREBP2 (Invitrogen), respectively. Rabbit monoclonal anti-Histone 3 (Cell Signaling Technology) was used to measure Histone 3 as a nuclear loading control for normalization. Immunoblots were analyzed on a LI-COR Biosciences Odyssey Infrared Imaging System.
The nuclear content of NF-κB RelA in RAW cells or SREBP2 in HEK 293T cells was determined by quantitative immunoblotting using rabbit polyclonal anti-NF-κB RelA (Abcam) or rabbit polyclonal anti–SREBP2 (Invitrogen), respectively. Rabbit monoclonal anti-Histone 3 (Cell Signaling Technology) was used to measure Histone 3 as a nuclear loading control for normalization. Immunoblots were analyzed on a LI-COR Biosciences Odyssey Infrared Imaging System.