Sonicator ultrasonic processor

Protein extraction and assay were as previously described (33 (link), 34 (link), 64 (link), 65 (link)). Cells were lysed with radioimmunoprecipitation (RIPA) buffer (Sigma, Cat. # R0278) supplemented with protease and phosphatase inhibitor cocktails (Sigma, Cat. # P8340 and P0044, respectively). The lysates were sonicated twice for 30 seconds at 25% output power with a Sonicator ultrasonic processor (Misonix, Inc., Farmingdale, NY) and centrifuged (14,000 g; 4°C) for 30 min. Total protein levels were determined by the bicinchoninic acid assay (BCA, Thermo Fisher Scientific, Waltham, MA, USA, Cat.# 23228 and Cat.# 1859078).

786-O/VHL cells were crosslinked with 1% formaldehyde at room temperature for 10 min, and cells were pelleted and resuspended in 400 μl lysis buffer (1% sodium dodecyl sulfate, 10 mM ethylenediaminetetraacetic acid, 50 mM Tris–HCl, pH 8.0). Then DNA of the cells was sonicated and sheared to small fragments of 500–1,000 bp with sonicator ultrasonic processor (Misonix, Farmingdale, NY, USA). Subsequently, the supernatant of the sonicated cells was collected, diluted and precleared by protein A agarose (Santa Cruz Biotechnology). Furthermore, anti-VHL antibody (1:50, Novus biologicals, Littleton, CO) was added to the supernatant for immunoprecipitation with normal preimmuned mouse IgG (1:50, Santa Cruz Biotechnology) as a normal control. After overnight incubation, the protein A agarose were added and incubated for 3 h and then washed with low-salt, high-salt and LiCl buffers and the immunoprecipitated DNA was retrieved by 5 M NaCl at 65 °C for 4 h and purified with a PCR purification kit (TaKaRa). PCR was performed with specific primers for HNF-4α:5′-GGCAGCCTTATCTCTGCAAAAGC-3′ (Promoter, forward) and 5′-GTGGGGGTTAATGGTTAATC-3′ (Promoter, reverse), 5′-GGAGATGACTT GAGGCCTTACT-3′ (3′UTR, forward) and 5′-GGGGAATCGTTTCCAA GGCCTC-3′ (3′UTR, reverse), 5′-GGCTCTGACACTGCAGAGTTCTAGAAC-3′ (Enhancer, forward) and 5′-ACCAACTTACCCAGCTGCTAATCATTGC-3′ (Enhancer, reverse).