Anti flag m2 agarose beads

NCOA4-FLAG-HA (CTAP) plasmid was received as a gift from Prof. J Wade Harper (Harvard Medical School, Boston). LNCaP cells were transfected with NCOA4-FLAG plasmid for immunoprecipitation studies or kept as untransfected control (C). NCOA4-FLAG transfected cells were either treated with 10 nM R1881 (T) or kept as untreated control (C*). Following treatment, cells were washed with 3 mL PBS, harvested in ice-cold PBS, and pelleted using a refrigerated centrifuge. Cells were lysed with 750 μL 1X lysis buffer (Promega) containing protease and phosphatase inhibitor for 20 minutes on ice. After lysis, the lysates were centrifuged at 13200 × g at 4 °C, and 10 percent of supernatant was stored as input. The remaining supernatant was used for immunoprecipitation using anti-FLAG M2 agarose beads (Thermo Fisher) as per manufacturer’s instructions. Affinity separated FLAG-tagged protein was eluted using 35 μL IgG elution buffer (Thermo Fisher), denatured with an equal volume of Laemmli buffer, and stored at −80 °C before western blot analysis. Each experiment was repeated at least three times.

Co-immunoprecipitation was performed using anti-FLAG M2 agarose beads (Invitrogen) according to the manufacturer instructions. Briefly, HEK 293 cells were split at 1:12 into 15 cm plates and transfected with 8 µg of plasmid encoding TNFR1 (pCMV6-XL5-TNFR1) on day 1 by calcium phosphate transfection. On day 3, cells were lifted by shearing, washed once, and resuspended in ice-cold PBS at ~10⁶/mL. LTα and TNF were added at 12 and 25 ng/mL respectively, and the cells were incubated for 30 minutes at 4 °C while rocking. Cells were then washed 3 times in ice-cold PBS and resuspended in RIPA lysis buffer, then analyzed by Western blot. TNFR1 was detected using rabbit α-TNFR1 (Abcam, ab19139, 1:1000) and α-rabbit secondary, as described above. Full gels are shown in Supplementary Fig. 16.