Srebp2

Cells were harvested and lysed in buffer (50 mM Tris–HCl buffer, pH 7.6, 10 mM ethylenediaminetetraacetic acid, protease inhibitor cocktail (Roche, Basel, Switzerland)) and phosphatase inhibitor (Roche) containing 1% SDS. After sonication, each 100-μg sample of protein was subjected to SDS-PAGE (5–15% polyacrylamide gels, BIO CRAFT, Tokyo, Japan), with 2-ME, and separated proteins were transferred to PVDF. The membranes were incubated with primary antibodies, then by appropriate secondary antibodies, and they were finally visualized using ECL plus or ECL chemiluminescence (GE Healthcare, Chicago, IL, USA). The following primary antibodies were used in this assay: TDP-43 (Proteintech, #10782-1-AP, 1:1,000), β-actin (Sigma-Aldrich, #A5441, 1:5,000), SREBP2 (Cayman Chemical, #10007663, 1:200), LDL-R (Abcam, #ab30532, 1:100), SCAP (Protein tech, #12266-1-AP, 1:200), Insig-1 (Novus Biologicals, #NB110-55244, 1:500), S1P (Sigma-Aldrich, #HPA040702, 1:1,000) and S2P (Cell Signaling Technology, #2157S, 1:1,000).

To analyze the protein expression levels of LDLR, PCSK9, SREBP2, and HNF-1α, HepG2 cells were seeded and treated as described above, and the cells were washed with cold DPBS, harvested, and centrifuged (1200 rpm, 3 min). The collected pellet was lysed with the RIPA buffer (Elpis Biotech, Inc.) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). The lysates containing equal amounts of protein, were then loaded on Bis-Tris gels for electrophoresis (10%), before being transferred to nitrocellulose membranes. The blots were blocked in 5% skim milk solution for 1 h and probed with specific antibodies (PCSK9 (MBL Life Science, Woburn, MA, USA), LDLR (Biovision Inc., Milpitas, CA, USA), HNF-1α (Cell Signaling Technology, Inc., Danvers, MA, USA), SREBP2 (Cayman Chemical, Ann Arbor, MI, USA), and β-actin (Bethyl Laboratories Inc., Montgomery, TX, USA)]. After washing three times with tris-buffered saline (TBS) with Tween 20, the blots were incubated with peroxidase-conjugated purified goat anti-rabbit IgG (Enzo Life Sciences, Inc., Farmingdale, NY, USA) for 1 h. After an additional wash with TBS with Tween 20, the proteins were detected using chemiluminescence reagent (Pierce Biotechnology, Rockford, IL, USA). The chemiluminescent signal was analyzed using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).