Foxo1 c29h4

FFPE tissues were subjected to IHC using primary antibodies as follows: CD10 (56C6, Novocastra, Newcastle Upon Tyne, UK), BCL-6 (LN22, Novocastra), MUM1 (MUM1P, Dako, Glostrup, Denmark), GCET1 (polyclonal, Abcam, Cambridge, UK), FOXP1 (polyclonal, Abcam), FOXO1 (C29H4, Cell Signaling, Danvers, MA, USA) and Bim (Y36, Abcam) using the Ventana Benchmark XT automated staining system (Ventana Medical Systems, Tucson, AZ, USA). DLBCL cases were immunohistochemically sub-grouped into GCB or ABC phenotypes according to the Choi algorithms, as previously described [49 (link)]. FOXO1 expression was evaluated according to the staining intensity (0-3) and location (nuclear vs. cytoplasmic).

For Western blotting, cells were extracted in lysis buffer, resolved by SDS-PAGE and transferred to nitrocellulose membranes. Proteins were detected using specific primary antibodies directed against UGT1A (H-300, 1:200, Santa Cruz Biotechnology, USA), SIRT1 (H-300, 1:200, Santa Cruz Biotechnology, USA), Ac-FOXO1 (D-19, 1:200, Santa Cruz Biotechnology, USA), FOXO1 (C29H4, 1:1000, Cell Signaling, USA), TRAIL (C92B9, 1:1000, Cell Signal Technology, USA), Bim (C34C5, 1:1000, Cell Signal Technology, USA), FasL (1:200, BD pharmingen, USA) or BCL-6 (N-3, 1:200, Santa Cruz Biotechnology, USA). The protein expression levels were normalized with GAPDH (1:5000, shengxing, China). After washing with TBST, the membrane was incubated with HRP-conjugated secondary antibody (1:10000, KeyGen, China) for 1 h. The signal was detected by enhanced cheniluminescence (ECL, Millipore).

Western blot analysis was performed either on nuclear or cytoplasmic proteins extracted from liver samples by using a commercially available kit (CelLytic nuclear extraction kit, Sigma) according to manufacturer's protocol. Protein concentration in supernatants was measured at 560 nm using the BCA method [51 (link)]. Samples (80 μg and 30 μg for nuclear and cytoplasmic proteins, respectively) were prepared in Laemmli buffer, boiled at 95°C for 5′ then loaded into SDS-PAGE precast gels (Bio-Rad, Hercules, CA) and run under denaturing conditions. Proteins were transferred onto nitrocellulose membranes (GE Healthcare Life Sciences, Chicago, IL), blocked with 5% non-fat milk for 45′, followed by incubation with primary antibodies overnight at 4°C. Antibodies were as follows: p16 (sc1207), p21 (sc-471), p27 (sc-528), p53 (sc1311), SIRT1 (sc15404), HDAC1 (sc7872), C/EBPβ (sc150), (all from Santa Cruz, Santa Cruz, CA); 53BP1 (ab87097), Actin (ab8227) (all from Abcam, Cambridge, UK); FOXO1 (C29H4, Cell Signaling Technology, Danvers, MA). Membranes were washed and incubated for 2 h with the appropriate secondary antibody conjugated with HRP. Protein bands were detected using a chemiluminescent substrate (Bio-Rad) and imaged onto Kodak film.