V5 beads

Huh-7 cells were first infected with PRMT1 lentivirus for 48 h, and then transfected with V5-tagged YAP for an additional 24 hour, solubilized in IP buffer (Pierce), and immunoprecipitated with V5 beads (Sigma-Aldrich, A7345) overnight at 4 °C. The immunoprecipitants were washed three times with wash buffer (20 mM Tris [pH 8.0], 150 mM NaCl, 0.5% NP40, 0.5% TritonX-100, and 10% glycerol) and washed two times with PBS buffer. The proteins attached to the beads were subjected to trypsin digestion in a suitable buffer. Tryptic peptides were analyzed by mass spectrometry on a Q ExactiveTM HF-X mass spectrometer (Thermo Scientific).

For analyzing the in vitro interaction of VPS34 with UBE3C, recombinant HA-UBE3C was purified from baculovirus with anti-HA agarose (Sigma-Aldrich) and eluted with an HA peptide. Flag-VPS34 purified from baculovirus was immobilized on anti-Flag M2 beads and incubated with purified HA-UBE3C in the binding buffer (50 mM Tris [pH 7.5], 150 mM NaCl and 1% NP40) for 30 min. For testing the in vitro binding between TRABID and VPS34, His-VPS34 was purified from bacteria using Ni Sepharose and eluted by imidazole. V5-TRABID purified from 293T cells was immobilized on V5-beads (Sigma-Aldrich) and incubated with purified His-VPS34 in binding buffer for 30 min. In both cases, the beads were washed with binding buffer and the bound proteins were analyzed by western blot.

For immunoblotting, antibodies were purchased from Sigma-Aldrich (HRP-anti-Flag, Cat# A8592; anti-Myc-HRP, Cat# 16-213), Cell Signaling Technology (HRP-anti-rabbit IgG, Cat# 7074P2; HRP-anti-mouse IgG, Cat# 7076; anti-β-actin, Cat# 4967; anti-RIP1, Cat# 3493; anti-capsase8, Cat# 9746), ABclonal (anti-GAPDH, Cat# AC033; anti-HSPA8, Cat# A10898), Prosci (anti-mouse RIP3, Cat# 2283) and Abcam (anti-LAMP2A, ab125068; anti-V5-HRP, ab1325; anti-6×His, ab18184; anti-human p-MLKL, ab187091; anti-mouse p-MLKL, ab196436). Anti-human MLKL, anti-mouse MLKL and anti-human RIP3 antibodies are generated by our own laboratory.
Recombinant TNF was purified in the lab. ATP (#A600020-0005) was purchased from Sangon. Smac mimetics LCL161 (HY-15518), GSK872 (HY-101872), and NSA (HY-100573) were purchased from MedChemExpress. Trypsin (#25200072) was purchased from ThermoFisher Scientific. ThT (#596200), Flag beads (#M8823), Myc beads (#E6654), V5 beads (#A7345), Nec-1 (#N9037) phosphoenolpyruvate (#P7127) and pyruvate kinase (#P7768) were purchased from Sigma-Aldrich. Nec-1s (#2263) was purchased from BioVision. The TRIF_(677–698) proteins and Z-VAD-FMK were custom-made by WuXi AppTec. Recombinant RHIM-containing proteins (RIP1_(498–582), RIP3_(418–518), and ZBP1_(150–293)) and recombinant α-syn were generated previously.