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3 protocols using rabbit anti tet1

1

Western Blot Analysis of Protein Expression

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For Western blot analysis, total proteins were extracted from cell lines (1×106 cells/well) supplemented with protease inhibitors cocktail with protein extraction buffer (Novagen, Madison, WI, USA) with 2 × SDS lysis buffer. Protein concentrations were quantified using the BCA protein assay kit (TIANGEN, Beijing, China). Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane. Subsequently, membranes were blocked with 5% non-fat milk powder dry milk in Tris-buffered saline with Tween-20 (TBS-T; 0.1% Tween-20 in TBS) and incubated with primary antibodies including rabbit anti-TET1 (Abcam), anti-Bax (Abcam), anti-Bcl2 (Abcam) and mouse anti-GAPDH (Abcam), respectively each at a dilution of 1:2000 in 5% blocking buffer overnight at 4ºC. Then, the membranes were washed twice with TBS-T, and membranes were incubated with HRP-conjugated secondary antibodies (anti-mouse or anti-rabbit, Invitrogen) for 1 h at room temperature (RT). The target bands were visualized using the Chemiluminescence Kit, and the protein bands were quantified using ECL Super Signal (Pierce, USA).
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2

Immunofluorescence Staining of Tet1 and γH2AX

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Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, washed three times with PBS (5 min each time), permeabilized with 0.25% Triton X-100 for 10 min at room temperature, washed with PBS (three times, 5 min each), and blocked with 5% BSA for 60 min at 37 ℃. The cells were then incubated with the following primary antibodies: rabbit anti-Tet1 (1:500, Abcam) and rabbit anti-H2AX (1:400, Abcam), overnight at 4 ℃. Cells were washed with PBS and probed with Alexa Fluor 488-labeled donkey anti-rabbit IgG (1:1000; Invitrogen) for 60 min at 37 ℃. After three PBS washes (5 min each), the nuclei were stained with DAPI for 10 min. Five microscope fields (× 200) were selected randomly for the evaluation. The mean fluorescence intensity was analysed using the ImageJ software.
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3

Chromatin Immunoprecipitation and Methylation Analysis

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For ChIP experiments, cells were cross-linked with 1% paraformaldehyde for 15 minutes and Chromatins were sheared into an average 200-400 bp in length by sonication (Diagenode) and immunoprecipitated with following antibodies: rabbit anti-MeCP2 (Cell Signaling Technology), rabbit anti-NURR1 (Santa Cruz Biotechnology) and rabbit anti-TET1 (Abcam). Immunoprecipitated DNA fragments were collected by magnetic beads (Active Motif), purified, and subjected to real-time PCR using primers specific to regions spanning three NBREs on Th promoter. Data were normalized to values of the input DNA. For MeDIP and hMeDIP experiments, genomic DNA were extracted from cells, sheared, immunoprecipitated, collected and subjected to real-time PCR in a similar way as ChIP with following antibodies: mouse anti-5mC (Abcam) and rabbit anti-5hmC (Active Motif). The primers are listed in Supplemental Table 1.
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