Ethylene glycol bis succinimidyl succinate

Glutaraldehyde (25%, Polysciences Inc., Warrington, PA, USA) was diluted with water to a final concentration of 0.04% and added to an equal volume of a protein fraction. EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Thermo-Fisher Scientific, Rockford, IL, USA), a cell-impermeable, zero-length crosslinker was prepared just before use as a 200 mM solution in water. A cell-permeable EGS (ethylene glycol bis (succinimidyl succinate), Thermo-Fisher Scientific, Rockford, IL, USA) that forms a 12-atom cleavable spacer arm, was prepared as a 100 mM solution in DMSO, just before use.

ChIP assays were performed using the Chromatin Immunoprecipitation kit (Cell Signaling Technology, Danvers, MA). Mouse tongue or footpad epidermal layers were separated following Dispase II treatment, washed with PBS and cross-linked with 1% formaldehyde for 20 min. For β-catenin ChIP, tissues were cross-linked with ethylene glycol-bis(succinimidyl succinate) (Thermo Fisher Scientific) at 5 mM final concentration, with addition of formaldehyde to 1% final concentration after 30 min of incubation. All steps were performed at 4 °C unless otherwise indicated. Tissue was pulverized under liquid nitrogen with a mortar and pestle and lysed in ChIP buffer. Chromatin was sonicated to 200–900 bp using an S220 Focused-ultrasonicator (Covaris, Woburn, MA). Thirty microgram chromatin were incubated overnight at 4 °C with 10 μl anti-β-catenin antibody (Cell Signaling Technology, 9587P), anti-KLF4 (R&D Systems, AF-1329), anti-TCF3 (Active Motif, 61125) or control IgG, followed by addition of Protein G Magnetic Beads. Beads were rinsed in washing buffer and DNA analysed by qPCR. At least three biological replicates were analysed in each experiment, and three technical replicates were performed for each biological sample. Primers are listed in Supplementary Table 1.

To increase the rate of cell division, Giardia cells grown for 72 h were incubated for 4 to 5 h with fresh medium, as previously described (62 (link)). The harvested trophozoites were fixed on a slide with PBS containing 2% paraformaldehyde, 100 μM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma-Aldrich), and 100 μM ethylene glycol-bis(succinimidyl succinate) (Thermo Fisher Scientific) for 30 min at 37°C. The cells were then treated with 100 mM glycine in PBS for 5 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. After three washes with PBS, the slides were blocked with 3% BSA in PBS for 30 min, incubated with primary antibodies, and then treated with secondary antibodies. After staining, the slides were mounted and observed under a Zeiss LSM700 confocal microscope (Carl Zeiss).
The following antibodies were used at the indicated dilutions: anti-HA rat monoclonal antibodies (clone 3F10, Roche Applied Science; 1:100), anti-acetylated-α-tubulin mouse antibodies (clone 6-11B-1, T7451, Sigma-Aldrich; 1:800), anti-Myc mouse antibodies (Thermo Fisher Scientific; 1:50), anti-Glcentrin mouse antibodies (59 (link)), Alexa Fluor 555-conjugated donkey anti-rat IgG (Molecular Probes; 1:100), and Alexa Fluor 488-conjugated donkey anti-mouse IgG (Molecular Probes; 1:100).