Tu 02

Cell lysates were isolated by RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 1% Na-Doc, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche). Cell extracts were quantitated using a BCA protein assay kit (Thermo). Western blot analysis was performed standard techniques for Twist1 (Abcam, ab50887, 1:1000, Cambridge, MA), Prrx1 (Origene, TA803116, 1:1000, Rockville, MD, USA), TNC (Genetex, GTX62552, 1:1000, USA) and alpha-tubulin (Santa Cruz, TU-02, 1:3000, Dallas, TX). Protein detection for Western blotting was performed using ECL reagent (#1705061, Bio-Rad). The antibodies are described in Supplementary Table 1. Uncropped images of the gels are shown in Supplementary Fig. 11.

For Western blotting, protein samples were separated on 4–12% gradient Tris-Glycine or 12% Tris-Glycine SDS-PAGE Gels (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and transferred to PVDF membrane (Millipore, Billerica, MA, USA). Antibodies against tubulin (TU-02; Santa Cruz Biotechnology), PIM2 (MAB4355, R&D Systems, Minneapolis, MN, USA or HPA000285, Sigma-Aldrich), ETV6 (HPA000264, Sigma-Aldrich), β-actin (#4970; Cell Signaling Technologies, Danvers, MA, USA), XIAP (#14334; Cell Signaling Technologies), cleaved PARP-1 (#5625; Cell Signaling Technologies) and survivin/BIRC5 (#2808; Cell Signaling Technologies) were used. The BioRad ChemiDoc XRS Imager was used to capture signals from blots. We quantified each protein band using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized each target protein after background subtraction to its loading control (β-actin or tubulin).

To detect the close proximity of proteins Juno and CD9, Proximity Ligation Assay (PLA)–Duolink In Situ Red Starter Kit, DUO92101 (Sigma-Aldrich^®) was used as was described previously (Vondrakova et al., 2022 (link)). Fixed zona-free mouse mature oocytes (MII) were incubated overnight in 4°C with selected pair of primary antibodies: 1) experimental group: rat monoclonal anti CD9 (KMC8.8) (sc18869, Santa Cruz Biotechnology, Inc.) diluted 1:50 in 1% BSA and rabbit polyclonal anti Folate receptor 4 (Juno) (abx102438, Abbexa^®, UK) diluted 1:50 in 1% BSA; 2) positive control–proteins with known interaction: mouse monoclonal anti-α tubulin diluted 1:1000 in 1% BSA (TU02) (sc8035, Santa Cruz Biotechnology) and rabbit polyclonal anti-β tubulin diluted 1:1000 in 1% BSA (ab15568, Abcam, UK); 3) negative control–protein with known absence of interaction: anti-α tubulin and rat monoclonal anti-CD9 (KMC8.8) diluted 1:50 in 1% BSA. Washed oocytes were incubated with PLUS and MINUS PLA probes and amplified following the manufacturer’s protocol and transferred into 2 μL of VECTASHIELD Mounting Medium. Fluorescence was detected with a confocal microscope (Carl Zeiss LSM 880 NLO) (Imaging Methods Core Facility, BIOCEV, Vestec, Czech Republic).