Bax b 9

Immunoblotting was undertaken as previously described [25 (link),31 (link)], with the following mouse primary antibodies against the following human proteins: p53 (DO1 and 1801, a kind gift of Sir D.Lane); MDM4 (8C6 04-1555; Merck Millipore, Darmstadt, Germany); PARP-1 (33-3100; Zymed/Invitrogen^TM, Sunnyvale, CA, USA); and p27 (610241; BD Biosciences, San Jose, CA, USA). The following rabbit primary antibodies were also used: MDM2 (C-18; Santa Cruz, Buda, TX, USA; and 3G9 04-1530 from Merck Millipore for VCap alone); Cleaved caspase 3 (9664, Cell Signalling); SKP2 (ab68455, Abcam, Cambridge, UK); p21 (12D1, Cell Signalling); SLC7A11 (D2M7A, Cell Signaling), BAX (B9; Santa Cruz, TX, USA); β-actin (13E5, Cell Signalling); USP7 (A300-034A, Bethyl Laboratories); PARP (9542; Cell Signaling Technologies, for VCap alone); p73 (A300-126; Bethyl Laboratories, Montgomery, TX, USA) and Hsp60 (H-300, Santa Cruz). Primary antibody detection was undertaken using secondary polyclonal antibodies from goats (Invitrogen) that were horse radish peroxidase (HRP) conjugated and directed against immunoglobulins of mouse or rabbit, as appropriate.

Monoclonal antibodies against MED10 (C-2: #sc-393450), BAX (B-9: #sc-7480), BCL-xL (H-5: #sc-8392), Ki67 (#sc-23900), Vimentin (V9: #sc-6260), and SNAI1 (G-7: #sc-271977) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); OCT4A (#2840) and LIN28A (#8706) were purchased from Cell Signaling Technology, Inc. (CST, Beverly, MA, USA); and GAPDH (#sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The primers used in this study were listed in Supplementary Table S6. The pcDNA3.1-FOXP3, pcDNA3.1, Flag-pcDNA3.1-FOXP3, Flag-pcDNA3.1 were produced in our Lab. CS2-flag-Smad2 (Addgen plasmid #14042) and CS2-flag-Smad3 (Addgen plasmid #14052) was a gift from Joan Massague, pcDNA3-Flag-Smad4 (Addgen plasmid #80888) was a gift from Aristidis Moustakas, Pbv-Luc (Addgen plasmid #16539) and c-Myc promoter del-1 (Addgen plasmid #16601) was a gift from Bert Vogelstein. SiRNA-FOXP3 (sc-43569) and siRNA-Control (sc-43569) were from Santa Cruz Biotechnology. The primary antibodies for western blot were as follows: FOXP3 (150D/E4, Invitrogen, 1:1000), FOXP3Δ3 (16J4G6, Novus,1:1000), PCNA (PC10, Santa Cruz, 1:2000), Bax (B-9, Santa Cruz, 1:1000), Bcl2 (C-2, Santa Cruz, 1:1000), p53 (DO-1, Santa Cruz, 1:1000), Smad2 (A-11, Santa Cruz, 1:1000), Smad3 (38-Q, Santa Cruz, 1:1000), Smad2/3 (A-3, Santa Cruz, 1:1000), p-Smad2(s467, Abcam, 1:1000), p-Smad3(1D9, Santa Cruz, 1:1000), Smad4 (B-8, Santa Cruz, 1:1000), c-Myc (9E10, Santa Cruz, 1:1000), and GAPDH (6C5, Santa Cruz, 1:2000). The primary antibodies for immunohistochemistry were listed below: FOXP3 (150D/E4, Invitrogen, 1:100), FOXP3Δ3 (16J4G6, Novus,1:100), Ki-67 (Ki67, Santa Cruz, 1:50), c-Myc (9E10, Santa Cruz, 1:100), Smad2/3 (A-3, Santa Cruz, 1:100), and p-Smad3 (Santa Cruz, 1:100).