Excel spreadsheet of full, raw mass spectrometry results from coimmunoprecipitation (coIP) experiments targeting two Wolbachia proteins - WARP754 and WARP434. We sought to identify native interactions between Wolbachia WARPs and host proteins using coIP. Towards that end, we generated polyclonal antibodies against full-length WARP434 and WARP754 and used the resulting sera, and pre-immune bleeds as controls, for a coIP using Drosophila JW18 cells infected with Wolbachia wMel. For both WARPs, we were able to pull down the target WARP in bulk lysate for the experimental conditions and identified top host protein targets present across all replicates, and either not present in our negative controls or significantly diminished in coverage. Interestingly, WARP754 antibody was able to pull down WARP434 by coIP, suggesting a possible interaction between these two proteins. That said, the WARP434 antibody did not reciprocally pull down WARP754, so this result should be cautiously interpreted.

We used Protein A/G Magnetic Bead (Pierce, Thermo Scientific Catalog #88802), following the manufacturer's procedure for manual immunoprecipitation with slight modifications, as noted here. Initial bead preparation used 50µL of bead slurry per sample, washing as indicated. We added 50 µL whole anti-WARP754

sera of the antibody (or 50 µL pre-bleed whole sera, when applicable) in 450 µL binding buffer to the bead for 1 hour at room temperature with mixing, or at 4 °C overnight. For the cell lysate, we used JW18 cells with a native Wolbachia infection and cells cleared of the infection with Tetracycline. Cells were counted, washed twice in sterile PBS, and pelleted. Each pellet of 7.5 X 10⁶ cells was lysed by 5ml of lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.8, 10 % glycerol, 1 % Triton, 1 M Arginine, with Protease Inhibitor included (1 tablet per 10 ml of Thermo 88665), and held on ice for 10 minutes with periodic vortexing. Antibody prep was removed off the magnetized bead and replaced by 1 mL of lysate, and held at 4 °C overnight with rotational mixing. Bead was washed per protocol, transferred on last wash to fresh tube, and left on the bead for mass spec analysis. Bead was stored on 50 µL 25 mM Ammonium Bicarbonate solution.

For WARP434, our handling of the bead was identical to above protocol, but our optimization for WD0434 enrichment required fractionation of the samples before protein lysis. We followed Senichkin, VV et al. protocol for nuclear and cytoplasmic fractionation, specifically outlined in Appendix A (of the associated manuscript) for hypotonic and isotonic solutions, with the addition of NP40 for lysis as indicated before adding to the bead (prepared as above) for enrichment. Lysate was added to the bead for enrichment as above, this time each bead sample was split as nuclear and cytoplasmic fractions, soluble and insoluble (pellets) for each. Pellets of each fraction were lysed by lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.8, 10 % glycerol, 1 % Triton, with Halt Protease Inhibitor (1X) (Thermo Scientific #78430, without EDTA added) before bead addition. The washed bead, stored in 50 µL 25mM Ammonium Bicarbonate solution, was submitted for analysis to the Indiana University Laboratory for Biological Mass Spectrometry. Peptides from Drosophila melanogaster and Wolbachia pipientis were identified and analyzed by LC-MS on an Orbitrap Fusion Lumos Tribrid equipped with an Easy NanoLC 1200.