Hrp conjugated anti ha antibody

Proteins from saponin-lysed schizonts or supernatant and merozoite fractions from PIA assays, were separated on 4%–12% or 3%–8% acrylamide gels (NuPAGE, Invitrogen). Separated proteins were transferred to nitrocellulose membranes by electroblotting. Blots were probed with HRP-conjugated anti-HA antibody (Roche) 1:1000 or for two-step methods, a primary antibody was followed by HRP-conjugated secondary antibody (Millipore). Bands were detected using ECL Plus Western blotting reagent (GE Healthcare) and the ChemiDoc Imaging System (Biorad). Antibodies used in this study are described in Table S4.
To ensure low MW proteins less than 20 kDa as in Fig. 1f, remained on the nitrocellulose membrane during the blocking, antibody and washing steps, the membrane was subject to a post-blot MeOH fixation protocol^{43 (link)}. Briefly, after electroblotting, the membrane was placed in 50% MeOH for 30 min/4 °C, then 50% MeOH for 30 min/50 °C, before the usual Western blot procedure.

Deubiquitination activity of CYLD-CP5 was evaluated as below. One hundred nM of purified CYLD-CP5 or Venus-CP5 was mixed with 1 μM recombinant M1-linked tetra ubiquitin (R&D Systems) in 12 μL of reaction mixture containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT. The reaction mixtures were incubated at 30°C for 3 hours, then the reaction was terminated by boiling in SDS sample buffer. Degraded ubiquitin chain was visualized by SDS-PAGE and SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific).
In vitro self-ubiquitination assay of MARCH3 was conducted as follows. Five μL of purified MARCH3-CP5 or Venus-CP5 was mixed with 4 μM recombinant HA-ubiquitin (Boston Biochem) in 20 mM Tris-HCl (pH 7.5), 200 μM DTT, 5 mM MgCl2, 3 mM ATP, 40 nM recombinant E1 (Boston Biochem) and 300 nM recombinant UbcH6 (Enzo Life Sciences). The reaction mixture was incubated at 30°C for 3 hours. The reaction mixture was boiled in SDS sample buffer and subjected to SDS-PAGE followed by immunoblot analysis using HRP conjugated anti-HA antibody (Roche Life Science).

Total protein extracts were obtained from yeast cultures by trichloroacetic acid (TCA) preparation as described and were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto nitrocellulose membrane (33 (link)). Hemagglutinin (HA)-tagged Mec3 was visualized using a horseradish peroxidase (HRP)-conjugated anti-HA antibody (Roche, 3F10). Expression of genes cloned into the pBTM116 vector were detected with an anti-LexA antibody (Abcam, ab14553), and those cloned into the pACT2 vector were detected using an anti-HA antibody (Covance, 16B12). Myc-tagged Mcm10 was detected using an anti-Myc antibody (Thermo Scientific, 9E11), and endogenous Rad53 was detected using an anti-Rad53 antibody (gift from Dr J.F.X. Diffley).