S protein beads

The cells were lysed in NETN buffer (200 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.05% Nonidet P-40, 1 mM EDTA) containing protease and phosphatase inhibitors (GenDepot, Houston, TX, USA). To pull down SFB-tagged proteins, cell lysates were incubated with S-protein beads (Merck, Darmstadt, Germany). To pull down MYC-tagged protein (Aurora-A), the cell lysates were incubated with MYC-beads (Thermofisher, Waltham, MA, USA) or normal mouse lgG-conjugated beads (Santa Cruz, Dallas, TX, USA). After inverting at 4 °C overnight, the precipitated protein complexes were washed three times with NETN buffer and the bound proteins were eluted by boiling in 2X Laemmli buffer and vortexing for 30 s and subjected to immunoblotting with the indicated antibodies.

LNCaP cells stably expressing TRIM24-SFB were lysed in NETN (100 mM NaCl, 20 mM Tris-Cl, pH 8.0, 1 mM EDTA, and 0.5% [vol/vol] NP-40) buffer containing protease inhibitors for 20 min at 4 °C. Crude lysates were subjected to centrifugation at 21,100 × g for 30 min. Supernatants were then incubated with streptavidin-conjugated beads (GE Healthcare) for 4 h at 4 °C. The beads were washed three times with NETN buffer, and bounded proteins were eluted with NETN buffer containing 2 mg/ml biotin (Sigma-Aldrich) for 1 h twice at 4 °C. The elutes were incubated with S-protein beads (EMD Millipore) overnight at 4 °C. The beads were eluted with SDS sample buffer and subjected to SDS-PAGE. Protein bands were excised and subjected to mass spectrometry analysis.

At 48 h post-transfection, the cells were lysed using NETN buffer (200 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.05% Nonidet P-40, 1 mM EDTA) supplemented with protease and phosphatase inhibitors (GenDepot, Houston, TX, USA). For the isolation of SFB-tagged proteins, cell lysates were treated with S-protein beads (Merck, Darmstadt, Germany). Similarly, for the capture of MYC-tagged proteins, the cell lysates were exposed to MYC-beads (Thermofisher, Waltham, MA, USA). Following an overnight inversion at 4 °C, the precipitated protein complexes underwent triple washes with NETN buffer. The bound proteins were then eluted by boiling them with 2× Laemmli buffer and subjected to vortexing for 30 s. Subsequently, the eluates were subjected to immunoblotting using the specified antibodies.

Tandem affinity purification on chromatin was performed as previously described. RNF168 was subcloned into pMH-SFB (Addgene ID: 99391) to drive mammalian expression of SFB-tagged RNF168 proteins. Briefly, SFB-RNF168–transfected HEK293T cells were harvested with NETN buffer (150 mM NaCl, 0.5 mM EDTA, 20 mM Tris–HCl at pH 8.0, 0.5% NP-40) with protease inhibitors for 10 min at 4 °C. The supernatant was discarded, and the pellet was washed with NETN buffer and digested again with NETN buffer with Turbonuclease (Sigma-Aldrich) to obtain the chromatin-bound fraction for 1 h at 4 °C. After centrifugation, the chromatin cell lysate was incubated with Streptavidin Sepharose (GE Healthcare) overnight, followed by washing with NETN buffer three times and eluted with 2 mM biotin at 4 °C. The eluent was then incubated with S-protein beads (EMD Millipore) overnight, washed with NETN buffer three times, and eluted with 1 × Laemmli buffer. The immuno-complex was subjected to SDS-PAGE and excised for mass spectrometry analysis.

IPed samples were prepared as previously described (27 (link)). Briefly, HEK293T cells were transiently transfected with SFB-tagged ZMYM2. Cells were extracted with NETN buffer for 30 min at 4°C. Cell lysates were centrifuged, and the supernatants were collected as the soluble fraction. The pellets were digested with NETN buffer with Turbo-Nuclease and 1 mM MgCl₂ for 1 h at 4°C and collected as the chromatin fraction. The both fractions were incubated with streptavidin sepharose bead (GE healthcare) for 1 h at 4°C. The beads were washed twice with NETN buffer and eluted with 2 mM biotin at 4°C. The eluted supernatants were incubated with S-protein beads (EMD Millipore) overnight at 4°C and washed three times with NETN buffer. The protein mixtures were eluted by boiling with 1× sample buffer. The eluted complex was subjected to SDS-PAGE and MS analysis.