Protein Extraction and Western Blot Analysis

Protein was extracted using a lysis buffer consisting of (50 mM Tris–HCl (pH 7.4), 5 mM ethylenediaminetetraacetic acid, 250 mM NaCl, 50 mM NaF, 0.1% Triton X-100, 0.1 mM Na₃VO₄) supplemented with 1× protease inhibitor cocktail solution (Roche). Extracts were quantified using Qubit protein assay (ThermoFisher) and were run on polyacrylamide gels. Gels were transferred to polyvinylidene difluoride (PVDF) membranes using a wet transfer method in 3-(Cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer. Primary antibodies were incubated overnight at 4°C. The following primary antibodies were used in this study: anti-FLAG (A8592, Sigma), anti-TET1 (GTX124207, GeneTex), anti-TET1 (GT1462, Sigma), anti-5hmC (catalog # 39769, Active Motif) and anti-β-actin (A5316, Sigma). For the DNA slot blot analysis, we followed the protocol established previously with the exception of using a slot blot apparatus instead of a dot blot (19 (link)). Blots were imaged using FluorChem Q and unsaturated bands were quantified using the multiplex band analysis tool followed by normalizing to local background and β-actin.