Matrigel coated thermanox coverslips

Cells were lysed in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet-P40, 0.5% sodium deoxycholate and 0.5% SDS) containing protease inhibitor cocktail and 0.2 mM phenylmethanesulfonylfluoride (PMSF) (Sigma). Twenty micrograms proteins were resolved in SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membranes and then probed with primary and secondary antibodies before chemiluminescent substrate reaction and exposure onto CL-XPosure film. Signals were quantified with ImageJ software, normalised to loading controls and presented as relative levels to controls. For immunostaining, cells were seeded onto Matrigel-coated Thermanox coverslips (Thermo Fisher Scientific) and fixed with 4% paraformaldehyde before being stained with indicated antibodies as described previously [9 (link), 24 (link)]. The signal was visualised and captured with a Leica SP5 confocal microscope. Multiple images were captured and counted. Cells were stained as previously described [24 (link)] then analysed and sorted on Calibur, DIVA, or Aria flow cytometers (BD). All antibodies were with listed in Additional file 1: Table S2.

hESCs were split and cultured on Matrigel-coated Thermanox coverslips (Thermo Fisher Scientific) until the desired confluency was reached. Cells were then washed with Dulbecco's PBS (DPBS) and fixed for 10–20 min with 4% fresh paraformaldehyde solution. Excess paraformaldehyde was removed by washing three times with DPBS and the coverslips were then incubated with blocking/permeabilization buffer for 1 h followed by an overnight incubation with primary antibody at the appropriate dilution at 4 °C with tilting. The following day, coverslips were subjected to 10-min washes three times with DPBS, followed by a 40-min incubation with the appropriate fluorophore-conjugated secondary antibody in the dark. Coverslips were then subjected to two further 10 min washes with DPBS followed by a 10-min DPBS/4′,6 diamidino-2-phenylindole (DAPI) wash, with DAPI at a final concentration of 1 μg ml⁻¹ to counterstain the nuclei. All washes were carried out in dark. Slides were mounted onto microscope slides using Mowiol 4–88 solution and allowed to dry overnight. Slides were visualized using a Leica SP5 II confocal fluorescent microscope typically at × 64 magnification.