Silverquest kit

Immunoprecipitation (IP) was done to enrich for the antigen target of 2448. Lysate from IGROV1 cells were precleared with Protein G-Sepharose resin (GE Healthcare) and clarified by centrifugation. Samples were subsequently incubated overnight on a rotator with 2448-coated Protein G-Sepharose resin. After multiple washes with PBS and saline solution containing 150 mM NaCl at pH 7.4, elution was done with SDS-containing buffer. IP products were separated by SDS-PAGE and analyzed on Western blot or stained using SilverQuest kit (Invitrogen). Silver stained bands corresponding to the antigen of interest were manually excised and soaked overnight in 2.5 mM ammonium bicarbonate in 50% acetonitrile solution at 4° C. Samples were subjected to in-gel trypsin proteolysis and sent for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

To assess collagen type I deposition sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at p4, at a seeding density of 25,000 cells/cm², at day 4 and at day 10 of culture, as has been described previously (Capella-Monsonis et al., 2018 (link)). Briefly, at each time point media were aspired and cell layers were washed with HBSS. Subsequently, cell layers were digested with porcine gastric mucosa pepsin at a final concentration of 0.1 mg/mL in 0.05 M acetic acid (Fischer Scientific, Ireland) and incubated for 2 h at 37°C with gentle shaking. After digestion, cell layers were scraped and neutralised with 0.1 N sodium hydroxide. For electrophoresis, sample buffer (SDS, 1.25 M Tris HCl, glycerol, bromophenol blue) was added to the samples. Cell layers were analysed by SDS-PAGE under non-reducing conditions with a Mini-Protean® three electrophoresis system (Bio-Rad Laboratories, United Kingdom). Bovine collagen type I (125 μg/mL, Symatese Biomateriaux, France) was used as control on every gel. Protein bands were stained with the SilverQuest™ kit (Invitrogen) according to the manufacturer’s protocol. The gels were imaged with a HP PrecisionScan Pro scanner (HP, United Kingdom). Densitometric analysis of the α1 and α2 bands was performed with ImageJ software (NIH).

Cells were lysed in hypotonic buffer (10 mm Tris-HCl at pH 7.65, 1.5 mm MgCl2, 10 mm KCl) and disrupted by Dounce homogenizer. This extract was then centrifuged at 4° C to separate the cytosolic fraction from the pellet. The nuclear-soluble fraction was obtained by incubation of the pellet in high-salt buffer (to get a final NaCl concentration of 300 mM). Tagged UHRF1 was then immunoprecipitated with anti-Flag M2-agarose (Sigma), eluted with Flag peptide (0.5 mg/mL), further affinity-purified with anti-HA antibody-conjugated agarose (Sigma), and eluted with HA peptide (1 mg/mL). The HA and Flag peptides were first buffered with 50 mM Tris-Cl (pH 8.5), then diluted to 4 mg/mL in TGEN 150 buffer (20 mM Tris at pH 7.65, 150 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0 0.01% NP40), and stored at −20° C until use. Between each step, beads were washed in TGEN 150 buffer. Complexes were resolved by SDS-PAGE and stained using the Silver Quest kit (Invitrogen).