Anti flag

Human embryonic kidney cell lines (HEK293T from ATCC) were maintained in advanced DMEM, supplemented with Glutamine and 10% (v/v) fetal bovine serum. A day before transfection, approximately 3 x 10 6 cells were seeded on a 100 mm dish. Transfection was performed with a mixture of pcDNA plasmids expressing specific proteins, as indicated, using Lipofectamine 3000 (Thermo Fisher Scientific) with empty pcDNA vector plasmid as a balance. After 48 h, cells were harvested and lysed with sonication in the lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40, pH 7.5) with complete protease inhibitor mixture (Sigma). Some cell lysate was mixed with loading buffer and heated at 95 o C for 5 min. For immunoprecipitation, the cleared cell lysate was incubated with 20 µL of anti-FLAG magnetic beads (Sigma) with shaking at 4 °C for 5 hours. The beads were washed with the lysis buffer three times and bound proteins were eluted with FLAG peptides at 0.1 mg/mL. Samples were subjected to SDS-PAGE and Western Blotting analysis with appropriate antibodies. Antibodies used in the currernt study included anti-HA (COVANCE), anti-FLAG (Abnova), anti-DDB1 (Sigma), anti-Actin (Sigma), anti-DCAF1 (Santa Cruz), anti-SIRT7 (Santa Cruz), anti-Goat IgG (Santa Cruz), anti-Rabbit IgG (Sigma), and anti-Mouse IgG (Sigma).