Nupage sample reducing buffer

Protein was extracted from fetal heart tissue by homogenising in RIPA lysis and extraction buffer, followed by protein quantification using a Pierce BCA assay (both Thermo Fisher Scientific). Samples (20 μg protein) were added to NuPAGE Sample Reducing Buffer and NuPAGE LDS Sample Buffer (both Thermo Fisher Scientific) and denatured at 70°C for 10 min prior to electrophoresis on a NuPAGE Novex 4–12% Bis Tris gel in NuPAGE MES SDS running buffer (Thermo Fisher Scientific). Following protein transfer to a nitrocellulose membrane, non-specific binding was blocked by incubation in 5% blotting-grade blocker (BioRad) in TBS-T for 1 h. Membranes were incubated with primary antibodies: GR (1:400; G-5: sc-393232, Santa Cruz) and β-tubulin (1:1000; MAB3408, Merck MilliPore) overnight at 4°C. Membranes were then washed and incubated with secondary antibodies IRDye 800CW Goat anti-mouse IgG (1:10000; 926-32210, Licor Biosciences) and IRDye 680RD Goat anti-Mouse IgG (1:10,000; 925-68070, Licor Biosciences), followed by quantification of GR protein levels relative to β-tubulin using an Odyssey infrared imaging system (Licor Biosciences).

Protein was extracted from left ventricle (LV) tissue by homogenising in protein extraction buffer (25 mM HEPES, 68.5 mM NaCl, 0.5 mM MgCl₂, 0.5 mM CaCl₂, 5 mM NaF, 1 mM EDTA, 5 mM sodium pyrophosphate, 1% NP-40, 10% glycerol, 1× protease inhibitor cocktail tablet). After centrifugation, protein concentration was measured relative to bovine serum albumin (Protein assay standard II, Bio-Rad) using Bradford assay. Protein samples (30 μg) were added to NuPAGE LDS Sample Buffer and NUPAGE Sample Reducing Buffer (both Thermo Fisher Scientific). Samples were denatured, electrophoresed on a NuPAGE Novex 4–12% Bis Tris gel in NuPAGE MES SDS running buffer (Thermo Fisher Scientific), and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA in Tris-buffered saline for 1 h, and then incubated with primary antibodies against GR (1:400; M-20, Insight Biotechnology, Middlesex, UK) and β-actin (1:10,000; 4967S Cell Signalling Technologies) at 4°C overnight, washed and incubated with secondary antibodies: IRDye 800CW Goat anti-Rabbit IgG (1:10,000; 926-32211 Licor Biosciences, Cambridge, UK) and IRDye 800CW Goat anti-Mouse IgG (1:10,000; 926-32210 Licor Biosciences). An Odyssey infrared imaging system (Licor Biosciences) was used to measure GR protein levels relative to β-actin using Odyssey software.

SH3GL1‐BioID2‐HA and empty‐BioID2‐HA constructs were cloned into a lentiviral expression vector (pLenti‐Puro, Addgene) and used to create single‐cell cloned stable Ramos cell lines. Expression was tested by Western blot and correct localization confirmed by immunofluorescence using anti‐BioID2 antibody (SS GD1, Novus Biologicals). 1 × 10⁸ cells per sample were cultured in 1 μM biotin in full RPMI for 14 h. Cells were washed and lysed in RIPA buffer containing protease inhibitor cocktail. Protein concentration was determined using a BCA assay (Sigma) and biotinylated proteins were pulled down using a corresponding concentration of Dynabeads Streptavidin C1 (Thermo), as per manufacturer's instructions. Biotinylated fraction was eluted in NuPAGE Sample Reducing Buffer (SDS‐containing; Thermo), boiled at 95°C for 5 min and in‐gel digestion performed. Peptide extracts were resolved on an EASY‐Spray column (Thermo) using UltiMate 3000 RSLCnano System. Data acquisition was performed using an Orbitrap mass spectrometer (Thermo) controlled by Xcalibur software. Data analysis was performed using the MaxQuant bioinformatics suite, using LFQ (Label Free Quantification) algorithm.