Dtssp

Transfected HEK293 cell lysate was incubated with 500 μg of peptide F (Proteintech) or control peptide (Proteintech) overnight at 4°C. The sample was cross-linked with 2 mM DTSSP (Thermo Scientific) for 1 hour at room temperature. The sample was precleared by adding 10 μg of mouse immunoglobulin G and 20 μl of protein A/G agarose and incubated at 4°C for 2 hours. After centrifugation at 1000g for 5 min, the supernatant was incubated with 5 μg of anti-DDK antibodies (OriGene) overnight at 4°C. Protein A/G PLUS-Agarose beads (40 μl) (Santa Cruz Biotechnology) were added and incubated for 2 hours at 4°C, then centrifuged for 5 min at 1000g at 4°C. The pellet was washed three times with IP lysis buffer (Thermo Scientific), followed by addition of 80 μl of 2× sample buffer (Santa Cruz Biotechnology). The sample was boiled for 5 min and then centrifuged for 5 min at 1000g at 4°C. The supernatant was loaded onto a 15% SDS-PAGE gel and probed with antibodies to peptide F (nonreducing condition), DDK, and CD59 (both in reducing condition with 5% β-mercaptoethanol).

For GFP:DDI2 mass spectrometry, pellets from GFP:DDI2 or GFP expressing cells was resuspended in IP buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 2 mM MgCl2, 0.1% Tween-20) supplemented with protease inhibitors (Roche) and 2 mM DTSSP (Thermo), sonicated for 3×20 s at setting 15 (Misonix), and microcentrifugated at maximum speed. Crosslinker was quenched with addition of 20 uM Tris. Lysate was incubated with m270-Epoxy resin (Invitrogen) coupled to rabbit anti-GFP (in house) overnight at 4C. Resin was washed 6X with IP buffer, eluted in 0.5N NH4OH + 0.5mM EDTA, and eluate was dried down O/N in a vacufuge. Protein was resuspended in 1X Laemmli buffer, boiled for 30 min to reverse crosslinks, and run on a 4-12% Bis-Tris gel (Invitrogen) for in-gel digestion. For IP-western blot, cell pellets were resuspended in modified MCLB buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors and +/− 2mM DTSSP where indicated, and IPed as above. Resin was boiled for 30 min in 1X Laemmli buffer and run on a 4-12% Bis-Tris gel (Invitrogen) for immunoblotting.

Desolvation 3HA nanoclusters were generated as previously described with modification (21 (link)). In brief, the solution contained 1.6 mg/ml of protein in PBS and was desolvated with a 4:1 volume ratio of ethanol to protein solution. The particles were collected by centrifugation, and resuspended in sterile PBS with sonication. To coat an additional layer of 3HA molecules onto the 3HA nanoclusters, 800 μg of soluble 3HA protein was added at a concentration of 1.6 mg/ml to 480 μg desolvated 3HA nanoclusters and an amine crosslinking reaction was performed using 3 mM 3,3′-Dithiobis[sulfosuccinimidylpropionate (DTSSP, Thermo Scientific, Waltham, MA) for 12 hours while stirring to stabilize the nanoparticles. Coated nanoclusters were collected by centrifugation, and protein concentration was measured by a BCA assay according to the manufacturer’s instructions (Thermo Scientific) to estimate the total protein content in nanoparticles. Dynamic light scattering (DLS) was performed in PBS with a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) to assess nanoparticle size distributions. For scanning election microscopy studies, coated 3HA nanoparticles were resuspended in water, dried, and sputter coated with carbon prior to visualization with a Zeiss Ultra60 FE scanning electron microscope at 5.0 kV.