Anti flag hrp

NuPAGE precast gradient 4–12% polyacrylamide (Thermo Fisher Scientific) was used for anti-BirA blot. All other blots are either 10% or 12.5% polyacrylamide:bis (37.5:1) gels. The membranes were incubated in 5% milk in PBS with Tween or Nonidet P-40 at 0.2%. Anti-RHBDL4 (Sigma, HPA013972) was used at 1:200. Anti-K63 linkage–specific (Abcam, EPR8590-448), anti-p97 (Thermo Scientific Pierce), anti-mCherry (GeneTex, GTX128508), and anti-myc clone 4A6 (Merck Millipore, 05-724) antibodies were all used at 1:1000. Anti–FLAG-HRP (Sigma–Aldrich A8592) was used at 1:2000 whenever indicated. Neutravidin blots were done after blocking 3% BSA in PBS, with 0.2% Tween 20; neutravidin–HRP conjugate (Thermo Fisher Scientific) was used at 1:4000 in 3% BSA in PBS. 0.1% Ponceau S stain in 1% acetic acid was used to stain nitrocellulose membranes.

LUminescence-based Mammalian IntERactome (LUMIER) assay was carried out by transfecting HEK293 cells in 96-well plates with plasmids that expressed each client protein fused to NanoLuc luciferase (Promega) along with 3XFLAG-tagged HSP90α and HSP90β. After 18 hours, cells were washed with cold PBS and lysed with 100 μl TGNET complete buffer, then centrifuged at maximum speed for 15 minutes at 4°C. The resulting lysates were transferred to an anti-FLAG antibody-coated plate (Sigma) and incubated at 4°C for 2 hours with gentle shaking. The plates were washed with TGNET. Nano-Glo reagent (Promega) was added and luciferase activity was quantified using a plate reader (Perkin-Elmer). To normalize for 3XFLAG-HSP90 levels, the plates were washed again with TGNET and anti-FLAG-HRP (Sigma-Aldrich) was added. The plates were once again incubated at 4°C, washed and assayed for HRP luminescence activity. The experimental relative interaction strength for each client protein interaction was determined by dividing the NanoLuc readout by the HRP readout value. Each sample was assayed 3 times with 3 replicates. Standard deviations are represented by error bars. A two-tailed T-test was used to determine statistical significance. All calculations were performed in Excel (Microsoft).

For Western blot, protein samples were extracted from transfected S2 cells and analyzed by standard biochemical procedures. Primary antibodies used include: anti-α-tubulin (mouse, Sigma B5–1–2, 1:500000), anti-p-Ser-PKC (rabbit, Cell Signaling #2261 S, 1:300), anti-Flag-HRP (rabbit, Sigma A8592, 1:1000), anti-HA-HRP (rat, Roche 3F10, 1:1000), or anti-c-Myc-HRP (mouse, Santa Cruz Biotechnology sc-40 HRP, 1:500). For Co-IP analysis, protein lysates from S2 cells were harvested and lysed using the standard protocols. Flag antibody-conjugated beads (mouse, sigma A2220) or the anti-HA affinity matrix (rat, Roche 11815016001) were used to pull down the corresponding proteins for 2–2.5 hours at 4 °C. After serial washes with lysis buffer, resins were subjected to SDS-PAGE for analysis with antibodies described above. For experiments with drug treatments, drugs used were: MG132 (50 μM, BioVision), E64 (1 μM, Merck Millipore), OA (0.1 μM, Sigma 75320), and PMA (0.01 μM, Sigma P8139).
For the CIP dephosphorylation assay, transfected cells were lysed in lysis buffer (1% NP-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA, 14 mM β-mercaptoethanol, protease inhibitor, and 1 mM PMSF). Supernatants from cell lysates were incubated with or without CIP (New England Biolabs) at 37 °C for 40 min and analyzed by Western blot.

Antibodies used were anti-RIPK1 (BD Biosciences, San Jose, CA, USA, #610459), anti-actin (MP Biomedicals, Solon, OH, USA, #69100), anti-IκB-α (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-371), anti-hRIPK3 (ThermoFisher Scientific Pierce, Waltham, MA, USA, PA1-41533), anti-mRIPK3 (Sigma-Aldrich, St Louis, MO, USA, #R4277 and IMGENEX, San Diego, CA, USA,, IMG-5523-2), anti-hMLKL (Genetex, Irvine, CA, USA, GTX107538), anti-mMLKL (Millipore, Billerica, MA, USA, #MABC604 and Abgent, San Diego, CA, USA, AP14272b-ev), anti-phospho-hMLKL (Abcam, Milton, Cambridge, UK, #187091), anti-hFADD (BD Biosciences, 610399), anti-mFADD (Millipore, 05-486), anti-thiophosphate ester (Epitomics, Burlingame, CA, USA, #2686-1), anti-Braf (ThermoFisher scientific, MA5-15495), anti-HPK1 (Cell Signaling, Danvers, MA, USA, #4472), anti-Hsp90 (Santa Cruz Biotechnology, sc-7947), anti-p38MAPK (Cell Signaling, #9212), anti-ERK1/2 (Cell Signaling, #9102), anti-Flag-HRP (Sigma-Aldrich, St Louis, MO, USA, #A8592) and anti-tubulin-HRP (Abcam, #ab21058). Secondary antibodies used were HRP-conjugated secondary antibodies against mouse, rabbit or rat immunoglobulin (GE Healthcare, Little Chalfont, Amersham, UK).