Sig 31043

Immunoblotting and co-immunoprecipitations were performed as described [48 (link)]. Antiserum against FOXO (C29H4) and anti-Flag M2 (F3165) was purchased from Cell signaling Technology and Sigma for immunoblot analysis. E2F1 antibody (C20) was purchased from Sigma. Anti-HA antibody was purchased from Roche (3 F10). Anti-p53 (sc-6243, Santa Cruz), p-FOXO1 (9461S, Cell Signaling Technology), p-Akt (sc-7985-R, Santa Cruz), Capase-3 (Cell Signaling Technology, Cat# 9661), Ki-67 (SP6) (Biocare Medical, Cat# CRM325), and TUNEL (EMD Millipore, Cat# S7101) were used for IHC. IHC detection was through primary antibodies listed in Additional file 1: Table S2, with Vector biotinylated secondary (1:250), tertiary was streptavidin-horseradish peroxidase (Covance #SIG-32000) and chromagen 3,3′-diaminobenzidine substrate (Covance #SIG-31043).

Retinoblastoma tissue microarrays were purchased from US Biomax (#BC35111a). IHC was performed as previously described [19 (link), 54 (link)]. Briefly, detection was through primary antibodies against p-AKT1, p-FOXO1, p-S6 & Ki-67 (SP6) (Biocare Medical, Cat# CRM325), using Vector biotinylated secondary (1:250), and streptavidin-horseradish peroxidase as tertiary (Covance #SIG-32000) and chromagen 3,3′-diaminobenzidine substrate (Covance #SIG-31043). TUNEL IHC analysis used the ApopTag Peroxidase kit from EMD Millipore (S7100). The University of Minnesota BioNet Resource assisted with H&E staining, KI-67 & TUNEL and other IHC staining as previously described [55 (link)]. All antisera used for IHC and immunoblotting are listed in Supplementary Table S2. Fisher's exact test was used to calculate two-tailed P-values for the contingency table comparisons.

TMAs were constructed from normal and neoplastic tissues collected at the University of Minnesota Medical Center. IHC was performed as previously described [39 (link)]. Briefly, detection was through primary antibodies against UHRF2 (Sigma HPA026697/HPA026633) (1:200), 5hmC (Active Motif F3165) (1:5000), & Ki-67 (SP6) (Biocare Medical, Cat# CRM325), with Vector biotinylated secondary (1:250), tertiary was streptavidin-horseradish peroxidase (Covance #SIG-32000) and chromagen 3, 3′-diaminobenzidine substrate (Covance #SIG-31043). Micrographs were taken at 400x magnification, except where noted.