Western blots

Whole limb buds from a single litter were lysed for 1 hr at 4C. For fractionation, 500,000 cells from limb buds were then dissociated with 100 ug/mL Liberase (Roche 05401119001), resuspended in CSKT buffer (10 mM PIPES pH6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 0.5% TritonX-100, incubated on ice for 10 min, and centrifuged for 5 min @ 5000 g. The cytoplasmic fraction (supernatant) and nuclear pellet were each resuspended in loading dye and boiled. Western blots were incubated with the following primary antibodies for 1 hr at room temperature in 3% milk: 1:4000 M2 Flag (Sigma 3165),1:4000 H3 (Cell Signaling 4499), 1:1000 GAPDH (Cell Signaling 5174), 1:1000 H3K27ac (Abcam Ab4729), 1:2000 B-actin (Cell Signaling 8457). Secondary antibodies were incubated for 1 hr at room temperature in 3% milk: 1:5000 Donkey anti-mouse (Jackson 715-035-150), Donkey anti-rabbit (Jackson 711-005-0152).