Ab10526

HeLa cells were grown to 60–70% confluency on 18 × 18 coverslips. The cells were treated with 20 mM 5-fluorouridine (5-FU) solved in DMEM for 3 min and then fixed 10 min with 2% formaldehyde. All washing steps after fixation were performed with 0.02% Tween in PBS (PBST). Cells were quenched in saturated glycin-solution and permeabilised with 0.5% Triton-X100 in PBS. Blocking was performed in 2% Bovine serum albumin (BSA) and 0,5% fish skin gelatin (FSG) in PBST. Primary antibodies used were: rabbit polyclonal anti JMJD6 (ab10526, Abcam), mouse monoclonal anti-BrdU (B8434, Sigma) and rabbit polyclonal anti-U2AF65 (ab37483, Abcam) Secondary antibodies (Jackson ImmunoResearch, Molecular Probes) were coupled to Alexa405 for blue fluorescence, Alexa488 for green fluorescence and Alexa594 for red fluorescence. Cells were post-fixed with 4% formaldehyde in PBS after incubation with secondary antibodies. After additional washing steps cells were counterstained with 200 ng/ml DAPI in PBST for 10 min. Cells were mounted on microscopy slides with Vectashield mounting medium (Vector Laboratories) and imaged using the DeltaVision OMX (Applied Precision).

For each immunoprecipitation experiment ca. 5 × 10⁷ HeLa cells were grown in DMEM (Biochrom) supplemented with 10% fetal calf serum, penicillin (100 U ml⁻¹) and streptomycin (100 μg ml⁻¹) at 37°C, 5% CO₂. Cells were harvested, lysed for 30 min on ice in lysis buffer (150 mM NaCl, 10 mM Tris–HCl pH 7.5, 0.5% NP-40) and then sonicated for 20 s. After centrifugation, the supernatant was incubated at 4°C for 2 h with 5 μg of anti-Jmjd6 antibody (ab10526, Abcam) and for control with non-specific rabbit IgG (Merck Millipore). The lysate–antibody complexes were then added to 50 μl of Protein G Sepharose beads (4 fast flow, GE Healthcare) and incubated for 2 h at 4°C. Following the immunoprecipitation, beads were washed four times with wash buffer 1 (150 mM NaCl, 20mM Tris–HCl pH 7.5) and four times with wash buffer 2 (300 mM NaCl, 20 mM Tris–HCl pH 7.5) and then resuspended in Laemmli buffer.

HeLa cells and human embryonic kidney (HEK) 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 U ml⁻¹) and streptomycin (100 μg ml⁻¹) at 37°C, 5% CO₂. For microscopy HeLa cells were grown to 50–70% confluence on 18 × 18 glass coverslips and transfected with expression constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 24 hours post-transfection, cells were fixed with 4% paraformaldehyde (15 min at room temperature) and permeabilised with 1% Triton-X-100 in phosphate buffered saline (PBS). JMJD6 (ab10526, Abcam), anti-BrdU (B8434, Sigma), anti-HA (H6908, Sigma) and anti-U2AF65 (ab37483, Abcam) were used as primary antibodies, Cy3-coupled anti-mouse (Jackson Immuno Research), Alexa647 anti-mouse (Invitrogen) and Alexa488 anti-rabbit (Invitrogen) were used as secondary antibodies.