Amersham protran premium 0.45 μm nitrocellulose membrane

For sample preparation Drosophila embryos were homogenized with a plastic pestle in sonication buffer (50 mM Tris-HCl pH7.9, 2 mM EDTA, 50 mM NaCl, 0.5 mM DTT, 10 mM Na-butyrate and 1x Protease inhibitor cocktail set I (Calbiochem)), then sonicated on “high” energy setting for four cycles of 30 sec ON/30 sec OFF in a Bioruptor sonicator (Diagenode). Homogenized samples were boiled for 10 minutes in 2x Laemmli sample buffer containing 5% β-mercaptoethanol, then centrifuged for 10 minutes at 13000 RPM. Sample supernatants containing 30 μg protein were separated by 10% Tris-Tricine-SDS PAGE and electrotransferred to Amersham Protran Premium 0.45 μm nitrocellulose membrane (GE Healthcare Life Sciences). Membranes were blocked in 5% nonfat milk and incubated with the following primary and secondary antibodies in the indicated dilutions: anti-acetyl-H4K5 (ab61236, Abcam, 1:500), anti-acetyl-H4K8 (ab15823, Abcam, 1:1000), anti-acetyl-H4K12 (ab61238, Abcam, 1:1000), anti-acetyl-H3K18 (ab1191, Abcam, 1:500), anti-acetyl-H3K23 (ab47813, Abcam, 1:1000) anti-H3 (ab1791, Abcam, 1:4000), goat-anti-rabbit IgG-HRP (P0448, Dako, 1:4000). Immunoblots were developed with Immobilon Western Chemiluminescent HRP substrate (Millipore) and recorded with a C-DiGit chemiluminescent blot scanner (Li-Cor Biosciences). Band intensities were quantitated with Image Studio software (Li-Cor Biosciences).

Nuclear extracts from DLBCL cell lines were prepared using commercial reagents (Affymetrix, High Wycombe, UK). Proteins were resolved by gradient SDS-PAGE (NuPAGE, Life Technologies) and transferred to an Amersham^™ Protran Premium 0.45 μm nitrocellulose membrane (GE Healthcare, Chalfont St Giles, UK). The membrane was blocked for 1 h with 5% fat-free milk powder in PBS, before incubation overnight with primary antibody, FOXP2-73A/8 antibody or JC12 (both used at a 1/30 dilution of ‘in house’ hybridoma supernatant). After washing, the membrane was incubated with 1/5000 dilution of horseradish peroxidase conjugated secondary antibody (Dako, Ely, UK). Labeling was detected using enhanced chemiluminescence reagent (GE Healthcare). Blots were re-probed with nucleophosmin (NA24, undiluted ‘in house’ hybridoma supernatant) to confirm adequate sample loading.