Nb100 2215

Hepatocytes were harvested and washed twice in ice-cold PBS. Total cellular protein was extracted using a protein extraction kit according to the manufacturer's instructions. Aliquots of 60 μg total protein were denatured for 5 min at 100°C and loaded on 10% sodium dodecyl sulfate polyacrylamide gels and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. After incubation with blocking buffer, membranes were exposed to polyclonal GRP78 (78 kD, 1:250, sc-376768; Santa Cruz, CA), SREBP-1c (68 kD, 1:1000; NB100-2215; Novus, USA), FAS (273 kD, 1:1000; C2065; Cell Signaling, Danvers, MA, USA), SORT1 (85 kD,1:1000; ab16640, Abcam, Cambridge, MA), P65 (65 kD, 1:1000; D14E12; Cell Signaling; Danvers, MA, USA), ACC1 (265 kD, 1:1000; ab45174, Abcam, Cambridge, MA), CHOP (27 kD, 1:1000; L63F7; Cell Signaling, Danvers, MA, USA), PCSK9 (65–80 kD, 1:1000; 85813, Cell Signaling; Danvers, MA, USA), SCD1 (37 kD, Cell Signaling; Danvers, MA, USA). After washing and incubation with secondary antibody, immunoreactive bands were detected by enhanced chemiluminescence (Beyotime, China) solution. The imprinting was exposed with X-ray film to radiograph the spectral band, and the gray value of the spectral band was measured with bandscan software version 5.0 (prozyme Inc, San Leandro, CA). Protein abundance is reported relative to that of β-actin as a ratio of optical densities.