Embryos (12–24 h) from BioTAP-N Br140 transgenic flies were collected and dechorionated by immersion in 50% bleach for 3 min, rinsed with distilled water, and dried. Embryos (0.2 g) were suspended in 150 µL of nuclear extraction buffer (10% sucrose, 20 mM HEPES at pH 7.6, 10 mM NaCl, 3mM MgCl2, 0.1% Triton X-100) supplemented with 0.1 mM PMSF in a 1.5-mL tube and homogenized on ice with a motorized pestle. Nuclear extraction buffer was added up to 1 mL, and the lysate was centrifuged at 2000g for 5 min at 4°C. The nuclear pellet was resuspended in 150 µL of nuclear extraction buffer, and homogenization and centrifugation steps were repeated. The final crude nuclear pellet was resuspended in 600 µL of Novex Tris-glycine SDS sample buffer (Invitrogen), including NuPAGE sample-reducing agent (Invitrogen), and then boiled for 10 min. For S2 cells, ∼5 × 106 cells expressing BioTAP transgenes were harvested; washed with PBS; lysed with 200 μL of Novex Tris-glycine SDS sample buffer (Invitrogen), including NuPAGE sample-reducing agent (Invitrogen); and then boiled for 10 min.
Twenty microliters was used for each Western blot. PAP antibody (1:1000; Sigma, P1291) diluted in TBST + 5% milk was used to detect the Protein A epitope of the BioTAP tag, and Clarity Western ECL substrate (Bio-Rad) was used for detection.
Twenty microliters was used for each Western blot. PAP antibody (1:1000; Sigma, P1291) diluted in TBST + 5% milk was used to detect the Protein A epitope of the BioTAP tag, and Clarity Western ECL substrate (Bio-Rad) was used for detection.