Anti shp

Chenodeoxycholic acid (CDCA), GW4064 and anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Guggulsterone (Z form) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Anti-HIF-1α was purchased from BD Biosciences (Palo Alto, CA, USA). The anti-SHP, anti-HDAC1 and anti-14-3-3γ antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). We used the human cDNAs of SHP (human, NM_021969) for the transfection assays [34 (link)].

Frozen liver tissues were lysed in a RIPA buffer supplemented with cOmplete™ Mini Protease Inhibitor Cocktail (Roche) and phosphatase inhibitors (PhosSTOP EASYpack; Roche). Extracted proteins (60 μg) were resolved in SDS polyacrylamide gels, and then transferred onto a PVDF membrane using semi-dry transfer (Immobilon-P; Millipore). The following primary antibodies were used: anti-FGF19 (MAB969, R&D Systems; ab154185, Abcam), anti-FGFR4 (8562, Cell Signalling), anti-CYP7A1 (sc-25536, Santa Cruz), anti-CYP3A4 (sc-53850, Santa Cruz), anti-SHP (sc-15283, Santa Cruz), anti-FXR (sc-1204, Santa Cruz), anti-CAR (PP-N4111-00, R&D), and anti-OSTβ (sc-163192, Santa Cruz). Protein loading was normalized to GAPDH (sc-25778 + HRP; Santa Cruz), β-actin (sc-47778, Santa Cruz), and α/β-tubulin (#2148, Cell Signaling). The bands were visualized using the SuperSignal West Pico Chemiluminescent Detection System (Thermo Scientific). Image and densitometry analyses were performed using MicroChemi Imaging Systems and GelQuant software (DNR Bio-Imaging, Israel).

Hepatic and ileal proteins (40 μg) were electrophoresed on SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Anti-SHP, FGF15, FGF21, CYP7A1, CYP8B1, LKB1, AMPK, and β-actin (Santa Cruz, CA) antibodies were used for detection the specific proteins.