Anti c ebpα antibody

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and newborn calf serum (BCS) were purchased from Gibco Life Technologies (Grand Island, NY, USA), Insulin, indomethacin, dexamethasone, 3-isobutyl-1methylxanthine, and Oil Red O solution were purchased from Sigma-Aldrich (St Louis, MO, USA). 3-(4,5)-dimethylthiazo-2-yl-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco (Solon, OH, USA). Primers were purchased from Macrogen (Seoul, Korea). Anti-C/EBPα antibody and anti-β-actin antibody were purchased from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Anti-PPARγ antibody and anti-adiponectin antibody were purchased from Abcam (Cambridge, UK).

293T cells (ATCC, CRL-3216) were transiently transfected with 4μg CMV or 4 μg CMV-C/EBPα in 100 mm dishes using 15 μL Lipofectamine 2000 (Invitrogen). Nuclear extracts were prepared two days later and gel shift assay performed using 1 ng of radio-labelled probe and 6 μg of nuclear extract, as described [24 (link)]. Oligonucleotide probes containing 5′-GATC or 5’-TCGA overhangs were radio-labeled to similar specific activity using Klenow enzyme and α-P³²-dCTP. Sense strands of the probes used, with binding sites underlined, were as follows:
Ebf1-α1: 5’-GATCCTGATAATGGAGGAAGAAATAAGCTAGCGG,
Ebf1-α2: 5’-GATCCAGTTTGCCTTTGAGTAATGTCGTCAATTTG,
Ebf1-α3: 5’-GATCGTTATTGTTAAAAGTTGGGCAAGGTTGAAATGC, and
NE-C/EBP: 5’-TCGAGGCCAGGATGGGGCAATACAACCCG.
Chromatin immunoprecipitation (ChIP) was conducted using 10 μL (1:50) rabbit monoclonal anti-C/EBPα antibody (Cell Signaling, #8178S) or normal rabbit immune globulin G (IgG, Cell Signaling #3900S) as described [25 (link)], with the use of the following genomic DNA PCR primer pairs:
Ebf1- α1F: 5’-CCCAGAAGTAAGGTGTACCAAGT,
Ebf1- α1R: 5’-CCAGCCTCCAGAGCAAAATC,
Ebf1- α2F: 5’-AGAGGCTCTTGCTATTTGAGCC,
Ebf1- α2R: 5’-ACTCAAGCCAAGTAACTCACCC,
Ebf1- α3F: 5’-GCGAGTTATTTGCAAAAGCGAA,
Ebf1- α3R: 5’-AGCTTTTGTACAAGCAGTTGGG,
PU.1enhF: 5’-CTGGTGGCAAGAGCGTTTC, and
PU.1enhR: 5’-CCACATCGGCAGCAGCAAG.