Anti tet1

The cells were collected by radio-immunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) containing 1x protease inhibitor cocktail solution(Roche). The total protein concentration was measured using the BCA protein assaykit(Bio-Rad). The proteins were separated by 10% SDS-PAGE and transferred onto PVDF membrane. Then, the membrane was blocked with 5% non-fat milk for 2h at room temperature, and then incubated at 4°C overnight with anti-TET1 (GeneTex) and anti-FLAG (Stratagene) antibody (1:1000 dilution), or mouse monoclonal anti-β-actin (Sigma) antibody (1:5000 dilution) for the internal control. Finally, the membrane was washed again and detected using ECL method (Thermo Fisher Scientific) by gel imaging system (Bio-Rad). The results were evaluated by the Image J software (NIH).

Formalin-fixed, paraffin-embedded specimens were from 88 patients diagnosed with UBC at the Department of Urology, Shanghai General Hospital, affiliated with Shanghai Jiaotong University, between 2007 and 2015. The ethics committees of the Shanghai General Hospital approved the protocol. Paraffin sections were deparaffinized in xylene for antigen retrieval, followed by the incubation with anti-TET1 (1:500, Cat# 124207; GeneTex, CA), anti-5 hmC (1:1,000, Cat# 39769; Active Motif, Carlsbad, CA), anti-AJAP1 (1:250, Cat# 223117; Abcam, Cambridge, UK), anti-β-catenin (1:500, Cat# 610153; BD Biosciences, San Jose, CA) antibody at 4°C overnight. On the next day, the 3,3′-diaminobenzidine (DAB) kit (DAB-0031; Maixin Bio, Fujian, China) was applied to visualize the localization of the antigen and counterstain sections with hematoxylin. Antibodies are listed in Table S2. Briefly, staining score of each slide was evaluated through staining intensity (0 = no staining; 1 = weak staining; 2 = moderate staining; 3 = intense staining) and percentage of positive tumor cells (1 = 0–25%; 2 = 25–49%; 3 = 50–75%; 4 = 75–100%). A final score was given by multiplying the staining with the intensity score between 0 and 12. A score of 0–6 signals low expression, whereas 7–12 indicates high expression.

Cytobuster Protein Extract Reagent (Merck Millipore, Billerica, MA, USA) complemented with protease inhibitor cocktail (Roche Diagnostics Scandinavia AB, Bromma, Sweden) used to prepare protein extracts. Rabbit polyclonal anti-TET1 (GTX124207, GeneTex Inc) and goat polyclonal anti-Actin (sc-1616, Santa Cruz Biotechnology) were used. Separate cytoplasmic and nuclear protein extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) with Halt Protease Inhibitor Single-Use Cocktail EDTA-free (Thermo Scientific) according to manufacturer’s instructions. Rabbit polyclonal anti-TET2 (21207–1-AP, Proteintech Group), mouse monoclonal anti-Lamin A/C (sc-376,248, Santa Cruz Biotechnology), and rabbit polyclonal anti-β tubulin (sc-9104, Santa Cruz Biotechnology) were used. After incubation with the proper secondary antibody, except for anti-Lamin A/C-HRP conjugated, bands were visualized using the enhanced chemiluminescence system (GE Healthcare).

The reprogrammed cells were harvested and lysed with cell lysis buffer (1% Triton X-100 in 50 mM Tris–HCl, pH 7.4 containing 150 mM NaCl, 2 mM Na₃VO₄, 100 mM NaF, and protease inhibitors). 10% of the cell lysate was used for Input. The rest of the lysate was incubated with antibody overnight at 4 °C and the target protein was captured by a 1:1 mixture of Ezview Red Protein A Affinity Gel (Sigma, P6486) and Ezview Red Protein GAffinity Gel (Sigma, E3403) for 4 h at 4 °C. The immunoprecipitated proteins were used to perform western blotting. The antibodies used in coimmunoprecipitation were anti-Sin3a (Abcam, ab3479), anti-Tet1 (Genetex, GTX124207), anti-HDAC1 (Santa Cruz Biotechnology, SC-8410), anti-Flag (CST, 14793s), and rabbit IgG (Millipore). rabbit IgG was used as a negative control.