Anti ha clone f 7

Anti-RHBDL2 and anti-E-cadherin antibodies were purchased from Proteintech (Rosemont, IL, USA). Anti-VE-cadherin (clone BV-9) and anti-HA (clone F-7) antibodies were supplied by Santa Cruz Biotechnology. EGFR phosphorylation was detected by a phospho-specific antibody (directed against p-Tyr¹⁰⁶⁸) from Abcam (Cambridge, UK). The total and phosphorylated forms of p44/42 MAPK(Erk1/2) and AKT (against pAKT-S⁴⁷³ and pMAPK-Thr²⁰²/Tyr²⁰⁴) were detected with antibodies from Cell Signaling Technology (Danvers, MA, USA). Other antibodies that were applied in this study were: for Western Blotting, anti-vinculin (Sigma), anti-N-cadherin (Abcam), anti-Thrombomodulin (D-3, Santa Cruz Biotechnology, Dallas, CA, USA), and anti-RFP (Rockland Immunochemicals Inc., Limerick, PA, USA), for immunofluorescence anti-E-cadherin (clone 36/E, BD Transduction Laboratories) and anti-VE-cadherin (clone F-8, Santa Cruz Biotechnology). The secondary antibodies were purchased from Jackson Laboratories (Bar Harbor, ME, USA). The E-cadherin function-blocking antibody DECMA-1 was purchased from Sigma Aldrich. Santa Cruz Biotechnology supplied the IKK inhibitor IKK-16, while the metalloproteases inhibitors BB94 and Marimastat and the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) were from Sigma. Human recombinant TNFα was purchased from Peprotech (Rocky Hill, NJ, USA).

Cell extracts from asynchronously growing cultures were prepared in EBX buffer (50 mM HEPES-KOH pH 7.5, 100 mM KCl, 2.5 mM MgCl₂, 10% glycerol, 0.25% Triton X-100, 0.5 mM TCEP, protease inhibitors, RNase and benzonase) using glass beads breakage in a cooled Multi-Beads Shocker (Yasui Kikai). Extracts were cleared by centrifugation, precleared, and incubated with either IgG coated Dynabeads (ThermoFisher) for Protein A pulldown or with Protein A Dynabeads previously coated with anti-Pk antibody. Beads were washed three times with EBX buffer and once with EBX containing 300 mM KCl, then elution was carried out in SDS-PAGE loading buffer. Antibodies used for pulldown and immunoblotting were mouse monoclonal anti-HA clone F-7 (Santa Cruz Biotechnology sc-7392, used 1: 5,000), mouse monoclonal anti-HA HRP conjugated clone GG8-1F3.3.1 (Miltenyi Biotec 130-091-972, used 1: 2,000), mouse monoclonal anti-V5(Pk) clone SV5-Pk1 (BioRad MCA1360, used 1: 2,000), mouse monoclonal anti-AID antibody (2B Scientific CAC-APC004AM, used 1: 5,000) and rabbit polyclonal anti-Sth1 antibody (a kind gift from B. Cairns, used 1: 5,000). Following incubation with a peroxidase-coupled secondary antibody, blots were developed using enhanced chemiluminescence reagents (Cytiva) and visualized using an Amersham 600 imager and its control software (v.1.2.0).

Chromatin immunoprecipitation was performed as previously described 79, as was the quantitative analysis of chromatin immunoprecipitates 80. Analysis of sister chromatid cohesion was done by visualising tetracycline repressor‐GFP fusion proteins bound to tetracycline operator arrays integrated at the URA3 locus on chromosome 5, as previously described 81. Hydroxyurea (HU) resistance was tested by spotting 10‐fold serial dilutions of cultured strains on YPD agar containing 0 M, 0.1 M or 0.2 M HU. Antibodies used for Western blotting or chromatin immunoprecipitation were anti‐HA (clone F‐7, Santa Cruz), anti‐tubulin (clone YOL1/34, Serotec) and an antibody specific to Rad53 (ab104232, Abcam).