Ab1791

Total proteins were extracted with Laemmli lysis buffer, sonicated (30 s ON/30 s OFF for 5 min) resolved on 10.5% acrylamide/bis-acrylamide SDS–PAGE gels and transferred to nitrocellulose membranes (Thermo Fisher Scientific) in transfer buffer. Protein transfer was assessed by Ponceau-red staining. Membranes were blocked in Tris-buffered saline pH 7.4 containing 0.1% Tween-20 and 5% milk for 1 h at room temperature. Incubations with primary antibodies were carried out at 4 °C overnight using the manufacturer recommended dilutions in Tris-buffered saline pH 7.4, 0.2% Tween-20. After 1 h incubation with an anti-rabbit or anti-mouse peroxidase-conjugated antibody (Jackson ImmunoResearch) at room temperature, proteins were detected by chemiluminescence using SuperSignal (Thermo Fisher Scientific), with Fusion v15.11 software or with the LI-COR Odyssey FC Imaging System and Image Studio Lite Software. The following antibodies were used: mouse anti-MyHC (R&D Systems, MAB4470), rabbit anti-histone H3 (Abcam, ab1791), rabbit anti-GAPDH (Sigma, G9545), monoclonal anti-Flag M2-Peroxidase (Sigma, A8592), rabbit anti-MCK (Kindly provided by Dr Slimane Ait-si-Ali, raised by Dr H Ito), rabbit anti-SMYD3 (Abcam, ab187149), rabbit anti-SMYD3 (Diagenode, C15410253-100), rabbit anti-Cyclin D1 (Abcam, ab 16663), rabbit anti-P21 (ab 109199), mouse anti-MYOGENIN (F5D ab 1835).

2 × 10⁵ cells were seeded one day before the experiments and washed twice with PBS before they were lysed in RIPA buffer. Protein concentrations were determined by BCA assay. Protein samples were resolved for SDS-PAGE, blotted onto PVDF membranes and probed with antibodies for PTGS1 (Sigma, AV41836), GPS2 (custom-made rabbit polyclonal IgG, as previously described (36 (link),39 (link)), NCOR (Bethyl, A301-145A), SMRT (Bethyl, A301-147A), H3 (Abcam, ab1791), STAT6 (Sigma, S-6433), p-STAT6 (Thermo, 700247), β-actin (Abcam, ab8226), H3K27ac (Abcam, ab4729), H3K4me3 (Abcam, ab8580), H3K9me2 (Abcam, ab1220), H3K9me3 (Abcam, ab8898) or KDM1A (Abcam, ab17721). Membranes were developed using the ECL system. All antibodies are listed in the Supplementary Table II.

For protein samples, 10–20 embryos were dechorionated manually and allowed to develop to the desired stage. At the appropriate time point, embryos were manually deyolked and snap frozen in liquid nitrogen. Cells were lysed in RIPA buffer containing HALT protease inhibitors for 20 min on ice and cell lysates were then centrifuged for 20 min at 20,000 × g. Supernatant was used for western blot analysis. Samples were run on 12, 18, or 4–20% acrylamide gels. For all blots, α-tubulin was used on the same membrane as a loading control. All western blots are representative of at least three biological replicates. Antibodies used include: anti-H3K9me3 antibody (Abcam ab8898, 1:1000), anti-H3 antibody (Abcam, ab1791, 1:5000), anti-α-tubulin (Sigma T6074, 1:2000). H3 histone bands were confirmed to run at 17 kDa and a tubulin to run at 55 kDa using PageRuler prestained ladder (Thermo Fisher). Western blots in the figures have been cropped to include only the relevant region, full uncropped blots are available as a Source Data file.