Anti flag m2 affinity beads

For preparation of lysates for immunoprecipitation (IP), cells were washed three times in ice cold phosphate-buffered saline (PBS) and lysed in 0.1% NP40 Lysis buffer (150mM NaCl, 20mM Tris pH 7.5, 0.5mM Ethylenediaminetetraacetic acid (EDTA), 1mM Na₃VO₄, 50mM NaF, 1mM β glycero-phosphate, 100 μM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml Leupeptin, 10 μg/ml Aprotinin). Cells were sonicated twice at 25% amplitude for 5 s with a thin probe, and lysates cleared by centrifugation. Where appropriate, antibodies were added at a concentration of 1 μg/mg of lysate and incubated overnight at 4°C, followed by antibody-protein complex capture with Protein G Sepharose beads (GE Healthcare) for at least one hour at 4°C. Alternatively, lysates were directly incubated with anti-Flag M2-affinity beads (Sigma Aldrich). After extensive washing in NP40 lysis buffer, complexes were eluted and analyzed by SDS-PAGE and immunoblotting.

Freshly egressed parasites collected from 20 T180 flasks (1.3–1.5 × 10¹⁰ tachyzoites) were washed as described above and resuspended in 5 mL PBS supplemented with TX-100 1% (v/v). All lysis steps were performed at 4 °C in presence of antiproteases (Complete, Roche) and 1 mM PMSF. A mechanical lysis step was performed with Dounce homogenization of parasites followed by 1 h incubation on rotating shaker. The insoluble fraction collected after centrifugation (16,000× g for 20 min) was resuspended in lysis buffer (1 × 10⁹ tachyzoites/mL) supplemented with 1% Empigen BB (Sigma-Aldrich) and 1 µL Benzonase (Sigma-Aldrich). This BCC7 enriched fraction was further submitted to a second step of mechanical Dounce homogenization and 1 h incubation on rotating shaker. The soluble lysate obtained after centrifugation (20,000× g for 45 min) was diluted half in lysis buffer without detergent and incubated with 1 mL anti-FLAG M2 affinity beads (Sigma-Aldrich). After 1 h incubation, unbound material was washed away from the beads with 30 mL PBS and elution was carried out with 150 mg/mL FLAG peptide. The presence of HA-FLAG-tagged proteins in the elution fractions was assessed by immunoblot analysis after SDS-PAGE. Experiments were conducted in triplicate with both RHΔku80_BCC7-HA-FLAG, and wild-type parasites were used as controls.

Parental COS-7 cells were purchased from ATCC (Manassas, VA). [³H]-labeled para-aminohippuric acid was purchased from PerkinElmer (Waltham, MA). Membrane-impermeable biotinylation reagent sulfo-NHS-SS-biotin, streptavidin agarose beads, protein G-agarose beads, and protease inhibitor cocktail were obtained from Pierce Biotechnology (Rockford, IL). Plasmid for wild type human Nedd4–2 was generously provided by Dr. Peter Snyder from University of Iowa (Iowa City, IA). Site-directed mutagenesis kit was purchased from Agilent Technologies (Santa Clara, CA). Mouse anti-myc and anti-β-actin antibodies were purchased from Roche (Indianapolis, IN). Mouse anti-E-cadherin antibody was purchased from Abcam (Cambridge, MA). Mouse anti-FLAG antibody and anti-FLAG M2 affinity beads were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit anti-phospho-Ser/Thr antibody was obtained from Cell Signaling Technology (Danvers, MA). Phorbol 12-myristate 13-acetate (PMA), staurosporine (St), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Human full length AAG and ∆80AAG lacking first 80 N-terminal amino acids were expressed and purified by using the established expression system^{57 (link)}. ELP1 cDNA was excised from pCMV6-XL5-ELP1 (OriGene) by NotI (NEB) and inserted into pcDNA3.1(+)-3xFLAG vector generated as described in^{8 (link)}. HEK293T cells were transfected by calcium phosphate, harvested after 48 h and WCE prepared as described above. 0.5 mg WCE were incubated with 75 µl anti-FLAG M2 affinity beads (Sigma Aldrich) for 2 h at 4 °C, the beads were washed three times with washing buffer (20 mM Hepes pH 7.9, 2 mM MgCl₂, 0.2 mM EGTA, 10% (v/v) glycerol, 0.1 mM PMSF, 2 mM DTT, 140 mM NaCl, 1x Halt™ Protease Inhibitor Cocktail). FLAG-ELP1 was eluted twice by addition of 80 µl Elution buffer (washing buffer with 0.5% (v/v) NP-40 and 0.15 µg µl⁻¹ 3x FLAG peptide (Sigma Aldrich)) for 30 min. The supernatant was applied on an Amicon Ultra-0.5 Centrifugal Filter Unit (Merck) and buffer exchanged to storage buffer (washing buffer with 0.1% (v/v) NP-40).