Pap antibody

Antibodies used in this study were as follows: α-Myc antibody (Myc; 9E10; Roche Applied Science, catalog no. 11667203001), PAP antibody (Sigma, catalog no. P1291), H3K4me1 (1:2000; Diagenode, pAB-037-050), H3K4me2 (1:1000; Diagenode, pAB-035-050), H3K4me3 (1:1000; Abcam, ab8580), H3 (1:2000; Abcam, ab1791), CTD Ser2 phosphorylation (1:1000; Abcam, ab5095), and CTD Ser5 phosphorylation (1:1000; Abcam, ab5131).

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.

Embryos of BioTAP transgenic flies were collected and dechorionated by immersion for 3 min in 50% bleach. The embryos were rinsed with distilled water and blot-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, 3 mM MgCl₂, 0.1% Triton X-100) with 0.1 mM PMSF in a 1.5-mL tube and homogenized on ice with a motorized pestle. Nuclear extraction buffer was added to the homogenate up to 1 mL and centrifuged at 2000g for 5 min at 4°C. Supernatant was removed, and the pellet was resuspended in 150 µL of nuclear extraction buffer. 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. Twenty microliters was used for each Western blot. PAP antibody (Sigma) against the Protein A epitope (1:1000 dilution) and Clarity Western ECL substrate (Bio-Rad) were used for the detection of BioTAP fusion proteins.