Purified mouse igg

3, 3′-dithiobis sulfosuccinimidylpropionate (DTSSP) and beta-D-Lactose were purchased from Thermo Scientific (Pittsburgh, PA). Sucrose was purchased from MP Biomedicals (Solon, OH). Ouabain octahydrate, cis-diammineplatinum dichloride (CDDP), doxorubicin hydrochloride (DXR) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Pyrazolo pyrimidine (PP2), a Src kinase inhibitor, was purchased from Tocris Bioscience (Bristol, UK).
Customized polyclonal rabbit anti-Gal-3 antibody was created by Pierce Biotechnology; mouse anti-V5 and purified mouse IgG were purchased from Invitrogen; polyclonal goat anti-Na⁺/K⁺-ATPase alpha1 and monoclonal mouse anti-Mdr-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal rabbit anti-Mdr was purchased from Oncogene Research Products (Cambridge, UK); mouse anti-beta-actin was purchased from Sigma-Aldrich; rabbit anti-Src and anti-p-Src were purchased from Cell Signaling Technology (Beverly, MA); purified rabbit IgG was purchased from ZYMED (San Francisco, CA); monoclonal mouse anti-phosphoserine was purchased from abcam (Cambridge, MA).

For immunoprecipitations, 0–2 h-embryos (≈100 μL embryos per IP) were homogenized in 500 μL DXB-150 (25 mM Hepes-KOH pH 6.8, 250 mM sucrose, 1 mM MgCl₂, 1 mM DTT, 150 mM NaCl, 0.1% Triton X-100) containing cOmplete^TM EDTA-free Protease Inhibitor Cocktail (Roche) and either RNase Inhibitor (0.25 U μL⁻¹, Promega) or RNase A (2 μg mL⁻¹, Sigma). A total of 50 μL Dynabeads protein A (Invitrogen) were incubated with either 10 μL mouse anti-GFP (monoclonal antibody 3E6, Invitrogen, A-11120), 5 µL mouse anti-Wisp (14D1, a gift from N. Kim), or 5 µL purified mouse IgG (Invitrogen, 02-6502) (mock IP) for 1 h on a wheel at room temperature. Protein extracts were cleared on 30 μL Dynabeads protein A previously equilibrated with DXB-150 for 30 min at 4 °C. The pre-cleared protein extracts were incubated with Dynabeads protein A bound to antibodies for 3 h at 4 °C. The beads were then washed 7 times with DXB-150 for 10 min at room temperature. Proteins were eluted in 1X NUPAGE buffer supplemented with 100 mM DTT at 70 °C and analyzed using western blots with antibodies at the following dilutions: mouse anti-Aub (4D10)^{48 (link)} 1:2500; rabbit anti-Aub (Abcam, ab17724) 1:2000; guinea pig anti-Wispy^{30 (link)} 1:3000; rabbit anti-Wispy^{31 (link)} 1:2500. Complete blots are shown in Supplementary Fig. 6.

Assays for total antibody were performed by coating 96 well flat bottom MaxiSorp plates (Thermo Scientific) with unlabeled goat anti-mouse IgA and IgG (Southern Biotech) (S1 Table) diluted in carbonate/bicarbonate buffer overnight at 4°C. Alternatively, plates were coated with 10⁷ CFU heat-killed S. boulardii resuspended in carbonate/bicarbonate buffer (5.4 mM Na₂CO₃, 8.6 mM NaHCO₃, pH 9.6) overnight at 4°C to detect antigen specific antibodies. Plates were blocked with TBST (150 mM NaCl, 15 mM Tris HCl, 4.6 mM Tris base, 0.5% Tween 20, pH 7.6) 5% nonfat dry milk for 2 hr at room temperature (RT) prior to incubation of serially diluted samples and standards overnight at 4°C. Goat anti-mouse IgA and IgG HRP-conjugated (Southern Biotech) antibodies were incubated for 1.5 hr at RT prior to addition of Super AquaBlue ELISA Substrate (eBiosciences) and reading at 405 nm. Anti-S. cerevisiae antibody (Abcam) and rabbit anti-goat IgG HRP-conjugated antibody (Southern Biotech) were used as positive controls for antigen specific assays. Purified mouse IgG (Invitrogen) and IgA (BD biosciences) antibodies were used as standards.