Ab52930

Samples collected from mice or cell culture were lysed by RIPA buffer containing protease inhibitor cocktail (04693132001, Roche) on ice. Equal amounts of protein were electrophoresed in 12% SDS-PAGE gel and transferred to PVDF membranes. These membranes were incubated with primary antibodies of HIP2 (ab52930, Abcam) and actin (m20010, Abmart) at 4 ℃ overnight, followed by secondary antibodies incubation at room temperature for 2 h. Images were obtained by Amersham (Imager 600, GE) and quantified by Quantity One (version 4.6.2, Bio-Rad).

Protein extracts were boiled for 10min in Laemmli buffer (10% (w/v) glycerol, 2% SDS, 10% (v/v) β-mercaptoethanol and 62.5 mM Tris–HCl, pH 6.8) and separated on a 4–12% SDS–PAGE followed by transfer onto nitrocellulose membranes. Prior to blocking the membrane for 1 h with 5% non-fat milk in TBST, membranes were briefly stained with 0.1% Ponceau-S in 5% acetic acid to represent total protein content. Membranes were subsequently probed with the indicated primary antibody in blocking solution for 16 h at 4°C: (E2-25K, 1:2000, ab52930, Abcam; GST, 1:500, ab9085, Abcam; HDAC1 was a kind gift from Dr, Alain Verreault; HIS-tag, 1:5000, 631212, Clontech; Histone H3, 1:1000, 9715, Cell Signaling; Histone H4, 1:1000, 2592, Cell Signaling; PML, 1:200, H-238, Santa Cruz; RAS, 1:1000, 3965, Cell Signaling; SP100, 1:300, ab43151, Abcam; SUMO2/3, 1:2000, 51–9100, Zymed; Tubulin, 1:1000, 2144, Cell Signaling; UBC9, 1:1000, 33044, Abcam; TRIM28/KAP-1, 1:200; A300-274A, Bethyl Laboratories.) The membranes were incubated with secondary antibodies (goat anti-rabbit HRP, AP307P, EMD Millipore, 1:5000 and goat anti-mouse HRP, AP308P, EMD Millipore, 1:5000) for 1 h. Membranes were washed three times with TBST. Membranes were revealed using ECL (GE healthcare) as per the manufacturer’s instructions, and chemiluminescence was captured on Blue Ray film (VWR) or with a BioRad ChemiDoc MP Imaging System.