Anti ha

Immunoprecipitation of SmHDAC8, SmDia-RBD and SmRho1 (isoforms and mutant constructs) proteins expressed in oocytes was performed using HA and Myc tags respectively. 15h after cRNA injection in the equatorial region, oocytes were lysed in buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 0.05% SDS, 0.5% Triton X100, 5 mM MgCl2, 1 mg mL^-1 bovine serum albumin, 10 mg mL^-1 leupeptin, 10 mg mL^-1 aprotinin, 10 mg mL^-1 soybean trypsin inhibitor, 10 mg mL^-1 benzamidine, 1 mM sodium vanadate) and centrifuged at 4°C for 15 min at 10 000 x G. Supernatants were incubated with anti-Myc (1/100; Invitrogen) and anti-HA (1/100 Invitrogen) antibodies for 4 h at 4°C. Protein A-Sepharose beads (5 mg, Amersham Biosciences) were added for 1 h at 4°C. Immune complexes were collected by centrifugation, rinsed three times, resuspended in Laemmli sample buffer, and subjected to a 10% SDS-PAGE. Immune complexes were analyzed by Western Blotting using anti-Myc (1/50 000, Sigma Aldrich) or anti-HA (1/10 000, Invitrogen) antibodies and the advanced ECL detection system (Amersham Biosciences).

The HA epitope was detected with mouse monoclonal antibody (mAb) HA.11 (BioLegend; 901515) and rabbit polyclonal antibody (pAb) anti-HA (Invitrogen; PI715500). The Ty1 epitope was detected with mouse mAb BB2 [61 (link)]. The c-Myc epitope was detected with mouse mAb 9E10 [62 (link)]. The V5 epitope was detected with mouse mAb anti-V5 (Invitrogen; R96025) and rabbit mAb anti-V5 (Cell Signaling Technology; 13202S). The OLLAS epitope was detected with rat mAb anti-OLLAS [63 (link)]. Toxoplasma-specific antibodies include rabbit pAb anti-IMC6 [64 (link)], mouse mAb anti-IMC1 [65 (link)], mouse mAb anti-ISP1 [32 (link)], rabbit pAb anti-Centrin1 (Kerafast; EBC004) [66 (link)], and rabbit anti-Catalase [67 (link)].

Recombinant full-length human RSPO2 was purchased from R&D Systems. RSPO2-Fu-WT and RSPO2-Fu-F109A were purified as described previously^{25 (link)}. For western blot analysis, anti-HA (Invitrogen cat #71–5500), anti-FLAG (Sigma cat # F7425), anti-β-actin (Cell Signaling cat #4970), anti-β-catenin (Cell Signaling cat #9562), anti-Axin1 (Cell Signaling cat #2087), anti-Axin2 (Cell Signaling cat #2151), anti-phosphor-ERK (Cell Signaling cat #9101), total ERK (Cell Signaling cat #4695), anti-PARP (cell signaling cat #9532), and anti-GAPDH (Cell Signaling cat #2118) were used. For ICC, anti-HA-Alexa 488 (Cell Signaling cat #2350), anti-human-Alexa 488 or –Alexa 555 (Invitrogen cat #A11013 and #A21433) were used. For western blotting, cells were lysed with RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1 mM DTT, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS), supplemented with protease and phosphatase inhibitors, and reduced at 37 °C for 1 hr. HRP-conjugated secondary rabbit or mouse antibodies (Cell Signaling) were used following the standard ECL protocol. For cytoplasmic β-catenin analysis, whole cell lysates were incubated with agarose bound concanavalin A beads to remove membrane-bound β-catenin and the remaining fractions were used as previously described^{40 (link)}.