Protease inhibitor cocktail 1x

Embryos were collected from conventionally reared flies and dechorionated using a bleach-based previously published method [59 (link)] to ensure that they were devoid of horizontally transmitted pathogens. One generation was then left to develop in conventional rearing conditions to allow for gut microbiota recontamination. Hemolymph was extracted from 10 days-old flies from the second generation after bleach treatment using a Nanoject II (Drummond) as previously described [16 (link)]. 1 μL of hemolymph was collected for each sample and frozen at -80°C until further use. Hemolymph was then diluted 10 times with PBS containing Protease Inhibitor Cocktail 1X (Roche). 1 μl was used for protein quantification with the Pierce BCA Protein Assay Kit (Thermofisher). The remaining 9 μl were mixed with SDS (0.2% final), DTT (2.5mM final) and PMSF (10μM final). Aliquots of 15 μg were used for proteomics analysis.

E12.5 embryos were dissected in PBS. Hearts were flash frozen and stored at -80°C during genotyping. 20 WT hearts and 20 Mesp1CreHira^fl/ hearts were pooled respectively and fixed for 15 min in 1% formaldehyde at 37°C then quenched by 125 mM of Glycine for 15 min at RT. Hearts were lysed in RIPA buffer (50 mM Tris pH7, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 50 mM NaF, 0.5% DOC, 0.1% SD. 2 μM sodium orthovanadate, protease inhibitor cocktail 1X (Roche) and PMSF 1 mM were added prior to the experiment. The hearts in lysis buffer were placed on a rotating wheel at 4°C overnight. A syringe (25G) was used to finish the lysis. The lysates were then sonicated (10 rounds of 1 min of sonication at 5 μA with Soniprep 150 MS, with 1 min on ice in between). The protein G beads were incubated overnight at 4°C in PBS with 10 μg of NKX2.5 antibody (N-19 Santa Cruz) and washed 3x in the previous RIPA buffer. They were then incubated with the sonicated chromatin at 4°C O/N. The following day, the beads were washed 2X for 5min in WB1 (10 mM HEPES pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and 2X for 5 min in WB2 (10 mM HEPES pH 7.6, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01% Triton X-100). The samples were then processed the same way as for HIRA qChIP.

Total protein extracts were obtained by lysing the cells in RIPA buffer (50 mM TRIS-HCl pH 7.4, 150 mM NaCl, 1% NP40, 1 mM sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM EDTA) with addition of Protease Inhibitor Cocktail 1x (Roche Applied Science, Penzberg, Germany). Cells were resuspended and centrifuged in the buffer and subsequently quantified by Bradford assay. A fixed amount of protein for each sample was loaded on SDS-Polyacrylamide gels for each sample and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in TBS + 0.1% Tween, and then immunoblotted overnight at 4 °C with the following primary antibodies: Tristetraprolin (#71632, Cell Signalling Technologies, Danvers, MA, USA); TWIST1 (#46702, Cell Signalling Technologies, Danvers, MA, USA); SLUG (#9585, Cell Signalling Technologies, Danvers, MA, USA); SNAI1 (#3895, Cell Signalling Technologies, Danvers, MA, USA). One-hour incubation at room temperature was enough for the primary antibody against CTNNB1 (BD Transduction Laboratories 610154, South San Francisco, California, USA). The nitrocellulose membranes were then incubated with secondary antibodies: either Goat anti-rabbit IgG-HRP or Goat anti-mouse IgG-HRP (Santa Cruz Biotechnology sc-2004 and sc-2005, Santa Cruz, CA, USA). Chemiluminescence was detected with ChemiDoc (BIO-RAD, Hercules, California, USA).

Vero cells were grown in 60-mm dishes and transfected with 1–2 µg of plasmid DNA using Lipofectamine (Invitrogen). 24 h pt cells were lysed with PBS supplemented with protease inhibitor cocktail 1x (Roche) by five cycles of freezing in liquid nitrogen and thawing at 37°C. Lysed cells were centrifuged at 300×g for 10 min and then at 30000×g for 20 min twice. Pellets were resuspended in the following solutions: 0.1 M Na₂CO₃, 4 M Urea (Merck), 1 M KCl (Merck), or 0.5% Triton X-100 (Sigma). Samples were boiled, resolved on SDS-PAGE and immunoblotted, following addition of Laemmli sample buffer.