Myc 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.

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.

The tagged NADK(Flag-NADK), Smurf1 (HA-Smurf1) and BMPR1A(Myc-BMPR1A) plasmids were transfected into HEK293T cells. 30h after transfection, HEK293T cells were treated with MG132 at a final concentration of 10μM for 4h, IP lysis buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1%NP-40, protease and phosphatase inhibitor) were used to harvest HEK293T cells. The supernatant was collected after centrifugation (12,000g, 15min). Myc-beads (Thermo Fisher, 88844) were added to the supernatant for incubation of 3h at 4°C. Then, the beads were washed 3 times in TBS buffer, mixed with 1×loading buffer added, and heated at 100°C for 10 min. Then, western blotting analysis was performed.