Anti ha magnetic beads

Freshly lysed parasites were harvested, filter-purified and centrifugated at 1000 × g for 10 minutes. Parasites were resuspended in 1 ml of PBS with 1% formaldehyde and incubate at room temperature with gentle agitation for 10 minutes. Parasites were centrifugated and resuspended with 0.125 M glycine solution in PBS and incubated at room temperature for 5 minutes to stop the cross-linking reaction. Parasites were centrifugated and washed with PBS one time and then resuspended with IP lysis buffer (25 Mm Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) and incubated at room temperature for 1 hour with rocking. The lysate was then centrifugated at maximum speed (~ 20,000 xg) for 10 minutes. The supernatant was collected and precleaned by incubation with 25 ul of mouse IgG magnetic beads (Cell signaling) for 1 hour at room temperature. The unbound lysate was isolated from the IgG beads by using a magnetic bead rack and were incubated with 25 ul of anti-HA magnetic beads (Fisher Scientific) for 1 hour at room temperature. Both mouse IgG (used as a control) and anti-HA magnetic beads were washed with IP lysis buffer for three times and with PBS for another three times.
For mass spectrometry analysis, samples were submitted to the Indiana University School of Medicine Proteomics Core facility for protein identification by mass spectrometry as described before⁵³ .

HEK293T cells (obtained and certified from ATCC) were cultured in DMEM (Invitrogen) containing 10% FBS. After transfection (Lipofectamine 2000, Invitrogen), cells were cultured for another 48 hr and then lysed with 2% TX-100 buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 2% TX-100, protease and phosphatase inhibitors (Gendepot)). After 10 min incubation on ice, lysates were centrifuged at 17,000 g, 10 min, 4°C, and supernatant was then used for immunoblotting or IP. For immunoblotting, sample buffer and reducing agent were mixed with each sample followed by a 10 min incubation at 70°C. Samples were then run on a 4–12% Bis-Tris gel, transferred to a PVDF membrane and blocked for one hour with 5% non-fat milk prior to primary antibody incubation. For IP, 20 µl antibody conjugated beads (anti-FLAG magnetic beads, Sigma-Aldrich, M8823 or anti-HA magnetic beads, Fisher, 88836) were added to the sample followed by overnight incubation at 4°C with rotation. Beads were then washed with 3 × 1000 µl of 0.2% TX-100 buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0.2% TX-100, protease inhibitors (Gendepot), and phosphatase inhibitor (Gendepot)) before being eluted in 2X elution buffer at 95°C for ten minutes.

As described previously (22 (link)), freshly lysed parasites were harvested and incubated for 10 min in PBS with 5 mM DSSO. Tris buffer was added to a final concentration of 20 mM to quench the reaction at room temperature for 5 min. After washes, the parasites were lysed with 1 mL RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail at 4°C for 1 h. The lysate was then centrifuged, and the supernatant was incubated with 25 μL of mouse IgG magnetic beads (Cell Signaling) for 1 h at room temperature for precleaning. The unbound lysate was separated from the IgG beads and was incubated with 25 μL of anti-HA magnetic beads (Fisher Scientific) for another hour at room temperature. The anti-HA magnetic beads were washed with RIPA lysis buffer and PBS and then submitted to the Indiana University School of Medicine Proteomics Core facility for mass spectrometry.

For co-immunoprecipitation (co-IP) experiments, cells were harvested at ~ 3.5-h post-meiotic induction (at the stage with maximum nuclear elongation), washed, resuspended in ice-cold Tris lysis buffer (100 mM Tris-Base, Tris-HCl, 1 M KCl, 1 M MgCl, 1% Triton X-100, 0.01 M PMSF, pH 7.5), and ground in a Dounce homogenizer. The cell lysate was clarified, filtered, and incubated with anti-HA magnetic beads (Thermo Fisher Scientific, Waltham, MA) for 2 h at 4 °C. (For details of the procedure, see Shodhan et al. 2017 (link).) After washing, two thirds of the protein-loaded beads were analyzed by mass spectrometry and protein eluted from the remaining third was analyzed by Western blotting.