Ab16408

Total cell extracts from freeze-clamped, pulverised tissues were obtained by incubation on ice for 10 min in cell lysis buffer (RIPA, Pierce) supplemented with proteinase inhibitors (1 × Complete (Roche), 1:500 PI cocktail (Sigma)) and 10 mM sodium butyrate (Sigma)). Lysates were sonicated (Bioruptor; 3 × 30 s, high setting) and spun at 13000 rpm, 4 °C for 30 s. Thirty microgram protein extracts were run on 4–20% Bis-Tris gradient gels (Invitrogen), transferred onto PVDF membranes (GE Healthcare) and stained with Ponceau S (Sigma). The blotted membranes were cut at ~40 KDa and blocked with 3% skimmed milk in TBS-T (TBS/0.02% Tween-20) for 1 h at room temperature. All antibody incubations were in 1% milk/TBS-T for 1 h at room temperature and washes between antibodies in TBS-T. The bottom half was incubated with rabbit anti-H3Ac (Millipore, 06-599, 1:10,000) and the top half with rabbit anti-LaminB1 (Abcam, ab16408, 1:1000). After 3 × 5 min washes, top and bottom half membranes were incubated with donkey anti-rabbit IR-Dye 800 (LI-COR, 926-32213), washed and scanned on a LI-COR Odyssey scanner. Fluorescence intensity signals were quantified with ImageQuant (GE Healthcare).