Autoimmune, Cholestatic and Biliary Disease
Version of Record online: 15 FEB 2013
Copyright © 2012 American Association for the Study of Liver Diseases
Volume 57, Issue 4, pages 1530–1541, April 2013
How to Cite
Chen, Y., Song, X., Valanejad, L., Vasilenko, A., More, V., Qiu, X., Chen, W., Lai, Y., Slitt, A., Stoner, M., Yan, B. and Deng, R. (2013), Bile salt export pump is dysregulated with altered farnesoid X receptor isoform expression in patients with hepatocellular carcinoma. Hepatology, 57: 1530–1541. doi: 10.1002/hep.26187
Potential conflict of interest: Nothing to report.
This work was supported by the National Institutes of Health (NIH) (grant no.: R01DK087755) and the NIH National Center for Research Resources (grant no.: P20-RR016457). B.Y. is supported by the NIH (grant nos.: R01GM61988 and R01ES07965). A.S. is supported by the NIH (grant nos.: R01ES016042 and K22ES013782). The authors thank Dr. David Mangelsdorf for providing the human FXRα1 expression plasmid.
- Issue online: 8 APR 2013
- Version of Record online: 15 FEB 2013
- Accepted manuscript online: 5 DEC 2012 02:51AM EST
- Manuscript Accepted: 1 NOV 2012
- Manuscript Revised: 30 OCT 2012
- Manuscript Received: 29 MAY 2012
Additional Supporting Information may be found in the online version of this article.
|HEP_26187_sm_SuppFig1.tif||2723K||Supporting Information Figure 1. FXR isoform expression detected by 2D gel and Western blot. Nuclear extracts from human liver tissues and HepG2 cells were prepared with the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce). Sample preparations for 2D gels were carried out with a ReadyPrep 2D cleanup kit and a ReadyPrep 2D starter kit (Bio-Rad). Electrophoresis was performed in strips with a range of isoelectric points from pH 5 to 8. For the second dimension, the gel strips were equilibrated and leveled on a precast SDS-PAGE gel (Bio-Rad), followed by Western blot using mouse anti-human FXR mAb (H00009971-M02, Abnova). The respective FXRα1 and FXRα2 spots were indicated by an arrow.|
|HEP_26187_sm_SuppFig2.tif||3257K||Supporting Information Figure 2. Detection of the signature peptides of FXRα1 and FXRα2 by mass spectrometry. Nuclear extracts from human liver tissues and HepG2 cells were reduced by 10mM dithiothreitol at 95 °C for 5 min in 25 mM ammonium bicarbonate buffer with 1% deoxycholate. The reduced proteins were then alkylated with 15 mM iodoacetamide in dark for 30 min. Trypsin was then added to each sample at a 20:1 protein: trypsin ratio, followed by digestion at 37 °C overnight with shaking. The digestion was terminated by adding equal volume of 0.2% formic acid. The digestion mixtures were dried in speedvac and the residue was reconstituted in 50 ul 0.1% formic acid solution. Twenty microliters of peptide solution were injected onto a fused-core C-18 column (Kinetex 100x2.1mm, 2.6mm, Phenomenex, Torrance, CA) by a CTC PAL autosampler (Leap Technologies, Carrboro, NC). A 24-minute gradient was delivered by a Shimadzu 20D HPLC system at a flow rate of 0.3 ml/min using the following gradient: 0min, 5% B; 17min, 45% B; 17.5min, 90% B; 20min, 90% B; 20.5min, 5% B; 24min, 5% B; in which mobile A is 100% water contains 0.1% formic acid and mobile B is 100% acetonitrile containing 0.1% formic acid. A TripleTOF 5600 mass spectrometer (AB Sciex, Foster City, CA) was used for data acquisition under information dependent acquisition (IDA) mode. A full mass TOF scan was performed first and the top 10 peptide-like precursor ions (m/z range 400-2000, +2 to +5 charges) were selected and fragmented to generate MS/MS spectra. ProteinPilot software (AB Sciex, Foster City, CA) was used to identify all peptide fragments from FXR using Paragon algorithm.|
|HEP_26187_sm_SuppTabs.doc||57K||Supporting Information Tables.|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.