P0217

Protein was extracted from cells using a lysis buffer (40 mM NaCl, 25 mM Tris pH 8, 2 mM MgCl₂, 0.05% SDS, 2x Complete EDTA-free protease inhibitor (Roche), 0.4 µL mL⁻¹ benzonase) with protein concentration determined by Bradford assay, comparing to BSA standards to ensure loading of an equal mass of protein into each lane. Protein and Laemmli buffer were heated to 100 °C for 5 min before loading into gel. For U2OS, NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen) was used. For MEFs, NuPAGE Novex 3–8% Tris-Acetate Gel (Invitrogen) was used. Samples were run on gels for 2 hr before transfer from gel to a nitrocellulose membrane. Antibodies used to bind proteins on membrane were pAb rabbit BRIP1/FANCJ antibody (1/10000, Novus Biologicals NBP1-31883), pAb rabbit RTEL1 antibody (1/5000, Novus Biologicals NBP2-22360). For loading control, mAb mouse anti-β-actin antibody (1/5000, Abcam ab8226) was used with U2OS and mAb mouse anti-α-Tubulin (1/10000, Sigma Aldrich T6199) was used with MEFs. Secondary antibodies used were: pAb swine anti-rabbit immunoglobulins/HRP (1/10000, Dako P0217) and pAb goat anti-mouse immunoglobulins/HRP (1/5000, invitrogen A16078). Visualisation was performed by exposure onto photographic film for U2OS and visualisation using Amersham ImageQuant 800 for MEFs. Full uncropped scans are available in the source data file.

HH29 and HH35 snap‐frozen hearts (n = 6 per treatment groups) were lysed using lysis buffer. The tissue was homogenised by sonication. Protein concentration was determined by Bradford protein assay (Sigma). GAPDH was used for normalisation.
The samples were run on an SDS‐PAGE gel with precision plus protein dual colour standards ladders (Bio‐Rad) transferred to a nitrocellulose membrane (Pall Corporation) and blocked using 5% BSA. Immunoblotting was performed using primary antibodies against TCF21 (1 : 750; sc377225; Santa Cruz), GAPDH (1 : 500; ab9485; Abcam), N‐cadherin (1 : 100; 6B3; DSHB) and E‐cadherin (1 : 25; 8C2; DSHB). The secondary antibodies used were horseradish peroxidase (HRP) conjugated rabbit anti‐mouse (1 : 2000; P0260; Dako) and swine anti‐rabbit (1 : 2000; P0217; Dako). Chemiluminescence was carried out using Amersham ECL Western Blotting Detection Reagents (GE Healthcare) and detected using photographic film (GE Healthcare). The photographic film was electronically scanned in TIF format. Both studies at HH29 and HH35 were repeated in triplicate. The analysis was carried out on fiji using the relative density of the pixels in each protein band, which was then normalised against GAPDH.

Whole cell lysate protein was extracted by using RIPA Lysis Buffer (Santa Cruz) and sonicated with Branson Snifier 150. The concentration of extracted protein was measured based on Bradford dye-binding method by using Bio-Rad Protein Assay Reagent and Bio-Rad SmartSpecTM 3000 spectrophotometer. Protein samples were fractionated by size on NuPAGE 4–12% Bis-Tris Gel (Novex) by electrophoresis and then transferred to Nitrocellulose Membrane (Protran, Whatman). The probed primary antibodies were detected by using Horseradish Peroxidise (HRP)-conjugated secondary antibodies and the Enhanced Chemiluminescent (ECL) detection system (GE Health). Primary antibodies: α-SMA (A5228, Sigma-Aldrich, 1:1000); SM22α (ab14106, Abcam, 1:1000); Calponin (EP798Y, Abcam, 1:1000); FSP-1 (ab27957, Abcam, 1:500); SOX2 (ab59776, Abcam, 1:500); OCT4 (sc-5279, Santa Cruz, 1:500); KLF4 (sc-20691, Santa Cruz, 1:500); CD144 (ab33168, Abcam, 1:500); CD31 (252253, ABBIOTEC, 1:500); Claudin 5 (352588, life technologies, 1:500); E-Cadherin (3195 P, Cell Signaling, 1:500); SNAI1 (3879 P, Cell Signaling, 1:1000); N-Cadherin (ab12221, Abcam, 1:300); HES5 (Ab5708, Millipore, 1:400); JAG1 (sc8303, Santa Cruz, 1:300); GAPDH (ab8245, Abcam, 1:2000). Secondary antibodies: anti-rabbit (P0217, DAKO, 1:3000); anti-mouse (P0260, DAKO, 1:3000).

Human urine samples were separated by 10 or 12% SDS-PAGE under reducing or non-reducing conditions as indicated. Proteins were transferred onto PVDF membrane (Millipore, Billerica, MA, USA) and probed with primary antibodies which detect either FSAP zymogen at 64 kDa or the light chain at 27 kDa (Mab677) or the heavy chain at 50 kDa (Mab1189). Bound antibodies were detected using horseradish peroxidase conjugated secondary antibodies (Swine anti rabbit polyclonal; P0217, Dako, Glostrup, Denmark) and the enhanced chemiluminescence detection system (Amersham-Pharmacia, GE Healthcare, Freiburg, Germany). Murine urine samples from mice were separated by 8% SDS-PAGE under non-reducing conditions. Proteins were blotted onto a nitrocellulose membrane (Amersham, UK) and probed with a primary antibody detecting FSAP zymogen at 64 kDa (Mab570). Bound antibodies were detected with a fluorescent secondary antibody labeled with IRDye 800CW and a fluorescence scanner (Licor Odyssey, Lincoln, USA).

Cell pellets were lysed with Cell Lytic M reagent (Sigma, MI, USA)) containing a protease inhibitor cocktail (Sigma, MI, USA). Proteins in crude lysates were quantified using the bicinchoninic acid assay (BCA) Protein Assay (Pierce Biotechnology, MA, USA)). A total of 20 μg of whole-cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Merck Millipore, Madrid, Spain). Blots were probed using primary antibodies (Table S3). Proteins were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-mouse (P0447, Dako, Barcelona, Spain) or anti-rabbit (P0217, Dako, Barcelona, Spain), incubated for 1 h at room temperature. Band intensity on the blots was quantified using the GeneTools Program (SynGene, Cambridge, UK).

FLAG-hscEMC10, HA-PKA and HA-AKT1 were all cloned into the lentiviral vector pLEX-MCS-CMV-puro (Addgene, USA). Transfection experiments were performed when the HEK293T cells were about 60–80% confluent, and cells were transfected with PEI reagents. Transfected 293T cells were lysed in EBC lysis buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40) supplemented with protease inhibitors (Selleck Chemicals) and phosphatase inhibitors (Selleck Chemicals). For immunoprecipitation, cell lysates were incubated with anti-FLAG M2 agarose beads or anti-HA agarose beads for 2 h. Beads were then washed four times with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40). Then the precipitated samples were separated by 10% SDS-PAGE gel and blotted with indicated primary antibodies. Primary antibodies used for western blot analysis were as follows: anti-Flag (1:3000; F7425; Sigma Aldrich), anti-HA (1:3000; SC-7392; Santa Cruz Biotechnology), anti-PKA C-α (1:1000; D38C6; CST). Peroxidase-labeled anti-mouse (1:5000; P0217; DAKO) or anti-rabbit (1:5000; P0260; DAKO) IgG secondary antibody was used.