Co ip buffer

Cells washed with ice‐cold PBS were first lysed with coIP buffer (Thermo Scientific). Then, the washed streptavidin‐coated magnetic beads (Invitrogen) were incubated with biotinylated in vitro‐transcribed circWDR37 or the corresponding complementary biotinylated RNA for 60 min. Subsequently, the RNA‐beads mixture was incubated with the prepared protein lysis buffer at 4 °C overnight. The next day, wash buffer was used to wash the beads for ten times. Finally, the collected proteins were used for mass spectrometry or western blot analysis.

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.

Co-immunoprecipitation studies were performed as previously described^{5 (link)} using anti-MYD88 antibody (Santa Cruz Biotechnology) and SYK, p-SYK(Y525/Y526) antibodies (Cell Signaling Technologies). Briefly, cells were lysed with Co-IP buffer (Thermo Fisher Scientific) supplemented with 1 mM sodium orthovanadate, 10 mM NaF, 1 × protease inhibitors cocktail for 15 min on ice, and then centrifuged at 2600 × g for 5 min. Supernatants (2 mg total protein) were incubated with 2–4 µg of antibodies at 4 °C for 30 min, followed by incubation with protein A/G-coated magnetic beads (EMD Millipore) for another 30 min at 4 °C. After samples were washed four times with ice-cold lysis buffer on a magnetic stand, proteins were eluted using SDS-PAGE loading buffer for further analysis.

MGC-803 or HEK293T cells were transfected with or without siRNA or plasmid for 48 hours, and 100 μg of protein extract was diluted to1 ml in Co-IP buffer (Thermo, USA); then, 2 μg of KIF23 (Santa Cruz Biotechnology, USA) or FLAG (Cell Signaling Technology, USA) antibody was added to the protein samples, and the mixtures were incubated overnight at 4°C with rotation. Twenty microliters of 50% protein A/G-agarose bead slurry (equilibrated in Co-IP buffer) was added, and after 2 h of incubation at 4°C, the beads were washed 4 times with Co-IP buffer (1 ml per wash) and diluted with 1×loading buffer. Western blotting was performed, and β-catenin (Santa Cruz Biotechnology, USA), Amer1 (Abcam, USA), and APC (Cell Signaling Technology, USA) were detected.