Anti hnf4α antibody

Nuclear extract were prepared as described previously [34 (link)]. Double-stranded biotin-labeled oligonucleotides (Invitrogen, Guangzhou, China) harboring the predicted HNF4α binding site on the Exo70 promoter (5′-CCTACTTAGCCCTTTGGACTACACAAGG-3′) were used as probes. Competitors were non-biotin-labeled oligonucleotides. The EMSA experiment was carried out using LightShift Chemiluminescent EMSA Kit (Thermo Scientific, cat. #20148) in accordance with the manufacturer's guidelines. For supershift, anti-HNF4α antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the nuclear extract after incubation with biotin-labeled probes. When the binding reaction finished, the mixtures were loaded onto a 6% polyacrylamide gel and subjected to electrophoresis, then transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Little Chalfont, UK). The biotin-labeled doublestranded DNA was then visualized using the Chemiluminescent Nucleic Acid Detection Module in this Kit.

Treated HepG2 and SMMC-7721 cells were collected and seeded onto glass coverslips processed for immunofluorescence. The glass coverslips were washed twice with cold PBS for 5 min, fixed with 4% paraformaldehyde for 20 min and incubated with 0.2%Triton X-100 for 5 min at 4 °C. After incubation, the cells were blocked with PBS containing 3% BSA for 1 h and incubated with anti-PKM1 antibody (1:200, Cell Signaling Technology) and anti-HNF-4α antibody (1:50, Santa Cruz Biotechnology) overnight. After being washed twice with cold PBS for 10 min, the cells were stained with FITC-conjugated anti-rabbit and anti-mouse IgG antibody (1:200, Jackson ImmunoResearch, Suffolk, UK) for 1 h. In addition, then the coverslips were stained with diamidinophenylindole (DAPI) for 30 min. The images were captured with a confocal microscope (Olympus FV1000, Tokyo, Japan).

The cells were fixed in PBS containing 4% paraformaldehyde (NJ-reagent, NanJing, CHINA) for 30 min and permeabilized with phosphate-buffered saline containing 0.1% Triton X-100 (NJ-reagent) for 20 min. The samples were incubated with anti-HNF4α antibody (1∶200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-human serum AFP antibody (1∶200; Santa Cruz Biotechnology), and anti-human serum ALB antibody (1∶200;Santa Cruz Biotechnology), and then with the secondary antibody conjugated to fluorescent phycobiliproteins, namely DyLight 594- and Alexa 488-conjugated goat anti-mouse immunoglobulin G (1∶1000; Beyotime, Shanghai, China). DAPI (Beyotime) was used for nuclear counterstaining.