Anti hnf 1 antibody sc 8986

Whole cell lysates prepared from mouse liver, Hepa1–6 cells and mouse primary hepatocytes, and 20 µg of protein were subjected to SDS-PAGE. The separated proteins were then immunoblotted as described previously using an anti-betatrophin (Angptl8) rabbit polyclonal antibody (no. 7619, ProSci, Poway, CA), an anti-HNF-1 antibody (sc-8986 ×, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), an anti-Phospho-Akt (Ser473) (D9E) antibody (#4060, Cell Signaling Technology., Danvers, MA), an anti-GAPDH antibody (ab9485, abcam, Cambridge, UK), an anti-cyclophilin A antibody (Upstate., Lake Placid, NY) and an anti-β actin antibody (#4967, Cell Signaling Technology., Danvers, MA).

Nuclear extract was prepared from Hepa1–6 cells after incubation with or without 100 nM insulin for 1 hr and subjected to immunoprecipitation. ChIP assays were performed using ChIP-IT Express kit (Active Motif, Carlsbad, CA) according to the manufacturer's protocol. Briefly, formaldehyde (37%) was directly added to the cell culture medium at a final concentration of 1% and incubated for 10 min. Cells were lysed and the nuclear lysate was sonicated three times with 15-sec pulses using a sonicator set at 25% of maximum power to yield the DNA fragments between 150 and 1000 base pairs in length. Sheared chromatin solution was immunoprecipitated with 3 μg of an anti-HNF-1 antibody (sc-8986 ×; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or preimmune mouse IgG (sc-2025; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For Ct value-based evaluation, qPCR was performed with power SYBR Green (Applied Biosystems Inc., Foster City, CA) using Applied Biosystems 7500 sequence detector with 1 μl of immunoprecipitate and input samples, according to the manufacturer's specified parameters. The primers for qPCR to amplify the region between −170 and +102 were as follows; sense: 5′-TAGCAGCGTGATATCAGCATTGC-3′ and antisense: 5′-TTGAGCTGGCTCTGGACCACCCAG-3′. The data represent %input ± SEM of tripricate PCR samples. ChIP assays were repeated twice.