Pureproteome protein a magnetic beads

Protein lysates (500 μg) were pre‐incubated with the antibody of interest (1 μg) for 2 hrs on a rotator at 4°C. Next, 20 μl of PureProteome Protein A magnetic beads (Millipore, St Charles, MO, USA) was added and incubated for additional 1 hr at 4°C. The immunoprecipitated complex was pulled down by the magnetic rack, washed with PBS/0.1% Tween 20 surfactant three times and eluted into 40‐μl electrophoresis buffer by heating at 90°C for 10 min. Western blotting was performed to analyse the components of IP complex.

CAFs were collected in lysis buffer (CHAPS 1%, TRIS-HCl 20 mM pH=7.5, NaCl 150 mM, EDTA 1 mM pH = 8.0, proteases and phosphatases inhibitors). Co-Immunoprecipitation was performed using PureProteome™ Protein A Magnetic Beads (Millipore, LSKMAGA10). Protein extracts were first precleared using 10 µl of beads for 200 µg of proteins (1-h incubation at 4 °C). 2 µl of anti-MCL-1 antibody (Cell signaling mAb #94296) and 10 µl of beads were then used for 200 µg of proteins following manufacturer’s instructions. For western blotting, DRP-1 antibody (MA5-26255, Thermo Fisher Scientific, Waltham, MA, USA) was used as primary antibody (1:1000) and the same anti-MCL-1 primary antibody (1:1000). Clean-Blot™ IP Detection Reagent (HRP) (Thermo Scientific #21230) was used as secondary antibody (1:1000). Whole uncropped images of the original western blots are presented in Supplementary Fig. 4.

For the SPANX-A/D interactome in human spermatozoa, cells were lysed using commercial Co-IP buffer (Thermo Scientific) with complete protease and phosphatase inhibitor cocktail (Roche) (quadruplicate). After protein homogenisation and sonication (20% amplitude, 10 pulses, three times), proteins were separated in soluble fractions by centrifugation at 13000 g and 4 °C for 15 min. The soluble protein fraction was incubated for 4 h at 4 °C with magnetic beads (PureProteome Protein A Magnetic Beads, Millipore) conjugated to an anti-SPANX antibody (ab119280, Abcam). This polyclonal commercial anti-SPANX antibody recognises an amino acid sequence (GDSDPQPAPKKMKTSE) corresponding to all SPANX-A, -B, -C and -D isoforms: As a negative control, rabbit IgGs (X0903, DAKO) were used at the same concentration as the antibody. Immune complexes were independently recovered and washed. Elution of the immunocomplexes was carried out with 8 M guanidinium hydrochloride at pH 8 and 70 °C for 15 min. The proteins were then reduced and alkylated, followed by in-solution digestion with LysC/trypsin. Peptides derived from each sample were concentrated and desalted using C18 stage tips (made in house using Empore Disc-C18 Agilent Life Science) for analysis by LC-MS/MS.

A549 cells were seeded at a density of 1×10⁵ cells/mL, and cultured for 24 h. After serum starvation for 24 h, cells were treated with TGF-β1 and KY-05009 for 48 h. After protein quantification, total lysates were pre-cleared with normal control IgG and PureProteome™ Protein A magnetic beads (Millipore). Immunoprecipitation of endogenous protein complexes was carried out overnight at 4°C with an anti-TCF4 antibody in combination with 30 µL of PureProteome™ Protein A magnetic beads. TCF4-bound proteins were subjected to Western blot analysis.

Per condition, 2 × 10⁶ cells were washed twice with phosphate-buffered saline (PBS) and serum-starved for 8 h at 37°C and 5% CO₂. Then, cells were either left untreated or incubated with 150 ng/ml HGF for 5 min. Cells were then washed twice with ice-cold PBS and lysed in 300 μl of lysis buffer [1% NP-40, 50 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM AEBSF, PhosSTOP (Roche Pharmaceuticals) and Complete Protease Inhibitor Cocktail (Roche Pharmaceuticals)]. Lysates were centrifuged at 15,000 g for 10 min at 4°C and immunoprecipitated with 0.7 μg of anti-phosphotyrosine antibody (4G10) overnight at 4°C. Immune complexes were captured with 50 μl of PureProteome Protein A magnetic beads (Millipore) at 4°C and washed three times with wash buffer (0.2% NP-40, 50 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM AEBSF, PhosSTOP, Complete Protease Inhibitor Cocktail). Immunoprecipitated proteins were eluted and boiled in Laemmli buffer.