Ab84497

Extracts for Western blotting were isolated as previously performed (Timms et al., 2016 (link)). Five million CD4⁺ T cells were lysed in cytoplasmic buffer containing 10 mM Hepes, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol, EDTA-free protease inhibitor cocktail, and 0.1% IGEPAL. The nuclear pellet was isolated by centrifugation at 1,500 rpm for 5 min. The total nuclear extract was isolated by incubation of the nuclear pellet with 1% SDS and 1:100 benzonase. The nuclear extract was heated in sample buffer containing SDS for 10 min at 70°C before separation with SDS-PAGE and transfer to a polyvinylidene fluoride membrane. The membrane was then blocked with 5% milk in TBST (Tris-buffered saline and Tween 20) followed by 1-h incubation with the primary antibody. The membrane was washed 4× for 5 min in Tris-buffered saline and Tween 20 before incubation in HRP-conjugated secondary antibody. The membrane was visualized with ECL reagent (Thermo Fisher Scientific). Antibodies used for Western blot were as follows: anti-MACF1 (ab84497; Abcam); rabbit anti-mouse anti-histone H3 antibody, Nuclear Loading Control and ChIP Grade (Ab1791; Abcam); rabbit anti-mouse anti-histone H3 (trimethyl K9) antibody, ChIP Grade (Ab8898; Abcam), peroxidase affinipure goat anti-rabbit IgG (H+L; 111-035-144; Jackson ImmunoResearch).