Ab4460

Total proteins were extracted using RIPA buffer (50 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) supplemental with protease inhibitors (Roche). KeyGEN Nuclear and Cytoplasmic Protein Extraction Kit (KGP150, KeyGEN BioTECH) was used to isolate nuclear proteins. Antibodies against MCM3 (ab4460, Abcam), p65 (ab16502, Abcam), p84 (ab487, Abcam), IKKβ (ab124957, Abcam), p-IKKβ (ab38515, Abcam), IκBα (ab32518, Abcam), p-IκBα (ab133462, Abcam), DNA PKcs (ab32566), DNA PKcs (phosphor S2056) (ab18192), CLEAVED PARP1 (ab32064) and GAPDH (G8795, Sigma).

Eighteen fresh tissue specimens of HCC and three fresh tissue of non-tumor adjacent tissue, as well as 162 paraffin-embedded HCC specimens were utilized, the detailed information was shown in Additional file 1: Table S1 The criteria for determining patient recurrence is that tumors is found in the liver, lung, skeleton, lymph and other positions after complete healing. These samples were collected during surgical procedures from patients with HCC according to a protocol approved by the institutional review board of the First Affiliated Hospital of Sun Yat-sen University. All patients provided written, informed consent for participation in the study and provision of tumor samples. IHC was performed according to our previous methods [23 (link), 24 (link)]. Anti-MCM3 antibody (ab4460, Abcam) was used. The images were captured using the AxioVision Rel.4.6 computerized image analysis system (Carl Zeiss Co Ltd., Jena, Germany).

Proteins (20–50 µg) were loaded on a polyacrylamide gel and then transferred onto Immobilon-P. Membranes were incubated with homemade rabbit polyclonal antibodies raised against recombinant full length 6His-human Ensa protein (anti-hEnsa) (1/1000), phospho-Arpp19/Ensa S67/S71 (Cell Signaling, #5240, 1/250,), human Greatwall (anti-hGreatwall) (1/1000), phospho-Greatwall S875^{34 (link)}, α-Tubulin (1/1000 clone C102, generous gift of J.M. Andreu, Spain), Treslin (1/1000, Bethyl Laboratories Inc., A303-472A), phospho-Ser CDK substrate (1/1000, Cell Signalling, #9477S), Histone H3 (1/500, Santa Cruz, Biotechnology Inc., SC-FL136), Cdk1 (1/1000, Santa Cruz, Biotechnology Inc., SC-954), MCM-2 (1/10000, Abcam, ab4461), MCM-3 (1/2500, Abcam, ab4460), MCM-4 (1/5000, Abcam, ab4459) and MCM-5 (1/5000, Abcam, ab17967). Mouse monoclonal used are MCM-7 antibody (1/1000, Abcam, ab2360), Flag M2 (1/500, Sigma, F3165), Vinculin (1/1000, Sigma,V9131 Hvin-1) and β-Tubulin (1/500, clone E7, generous gift from N. Morin). Uncropped scans of all the western blot membranes are supplied in the Supplementary Information file.