DOI: 10.5061/dryad.cfxpnvxms
title: Data from: <em>Wolbachia</em> uses ankyrin repeats to target specific fly proteins

description: 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.
description: 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
 <em>Wolbachia</em> 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 10<sup>6</sup> 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. <br> <br> 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 <em>Drosophila
 melanogaster</em> and <em>Wolbachia pipientis</em> were
 identified and analyzed by LC-MS on an Orbitrap Fusion Lumos Tribrid
 equipped with an Easy NanoLC 1200.
description: # Data from: *Wolbachia* uses ankyrin repeats to target specific fly
 proteins Dataset DOI:
 [10.5061/dryad.cfxpnvxms](https://doi.org/10.5061/dryad.cfxpnvxms) ##
 Description of the data and file structure Mass spectrometry after
 co-immunoprecipitation. ### Files and variables #### File:
 754coIP_supplementary_files.xlsx **Description:** Excel spreadsheet of
 mass spectrometry results for co-immunoprecipitation experiments using the
 anti754 antibody. A total of 17 experiments is included using both
 prebleeds and the antisera on JW18 cells infected with *Wolbachia* and
 uninfected, tetracycline-treated control cells. ##### Variables *
 Accession * Description * Coverage * Number of peptides * PSMs * Unique
 peptides * Area #### File: 434coIP_supplementary_files.xlsx
 **Description:** Excel spreadsheet of mass spectrometry results for
 co-immunoprecipitation experiments using the anti434 antibody. A total of
 16 experiments is included using fractionated JW18 cells infected with
 *Wolbachia*. ##### Variables * Accession * Description * Coverage * Number
 of peptides * PSMs * Unique peptides * Area #### File: movie_balancer.MOV
 **Description:** Video of fly behavior for sibling balancer flies not
 expressing WARP754. #### File: movie_754.mov **Description:** Video of fly
 behavior for flies expressing WARP754.