Wet blotting system

WB analysis was carried out as described by Nutzmann et al. [71 (link)]. Total protein (60 μg) was separated by SDS-PAGE and then transferred to PVDF membrane using a wet blotting system (Bio-Rad). The membrane was blocked with 5% BSA, incubated overnight with primary antibody (BxlB) and then gently shaken at room temperature following the addition of immunoglobulin G anti-rabbit secondary antibody labeled with peroxidase. Protein detection was carried out using the Clarity Western ECL Substrate chemiluminescence detection kit (Bio-Rad), as described by the manufacturer.

Total cell lysates (50 μg) were separated on 8, 12, or 14% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) Laemmli gels, depending on the molecular weight of the protein of interest. A wet-blotting system (Bio Rad) was used for protein transfer to nitrocellulose membrane, which were stained with 0.1% Ponceau (in 5% acetic acid) to evaluate transfer efficiency. Membranes were blocked for 1 h at RT in 5% non-fatty milk (in PBS). Primary antibodies were diluted in PBS with 5% BSA and 0.1% Tween 20. The antibodies were used at a concentration of 1:1000, except anti-tubulin that was used at 1:5000 as a loading control. Secondary Alexa Fluor-conjugated antibodies were diluted 1:5000 in 1% non-fatty milk. Membranes were analyzed using the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Total proteins extracted from the cells using RIPA lysis buffer (Millipore) were separated on 10% SDS‒PAGE gels (EpiZyme) and transferred onto polyvinylidene fluoride membranes using a wet blotting system (BIO-RAD). The membranes were blocked with 5% BSA in TBS-T, incubated with primary antibodies overnight at 4 °C, probed with HRP-conjugated secondary antibodies for 2 h at room temperature, and visualised using enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA). Information on the antibodies used is provided in Supplementary Table S3.

Membrane preparations (50 μg) were separated by SDS polyacrylamide gel electrophoresis (8% acrylamide-0.06% bisacrylamide) at constant current of 20 mA. The proteins were transferred onto nitrocellulose Hybond-ECL membranes (GE Healthcare Life Sciences, USA) using the wet blotting system (Bio-Rad, USA) for 90 min. Membranes were kept on PBS-Tween with 5% low-fat milk O/N and incubated with 5 mM diethyl pyrocarbonate (DEPC), to inhibit binding of S. aureus protein A to IgG [53 (link)] and rabbit polyclonal anti PBP2a antibody (raised against the synthetic peptide NH2-CGSKKFEKGMKKLGVGEDIPSDYPF; RayBiotech) at 1:1000 dilution for 1 hour. After two washes the membranes were incubated with the anti-rabbit secondary antibody conjugated to horseradish peroxidase (PerkinElmer, USA) at 1:5000 dilution for 1 hour. The chemiluminiscent signal was detected using Western Lightning Plus-ECL (PerkinElmer) and CL-XPosure film (Thermo Scientific). The membrane was incubated in stripping buffer (62.5 mM Tris-HCL pH 6.7, 100 mM β -mercaptoethanol, 2% SDS) at 50°C for 30 min and re-hybridized with 5mM DEPC and polyclonal antibody raised against the amidase domain of S. aureus Atl protein, at 1:1000 dilution for 5h.

Freshly egressed tachyzoites (1 × 10⁹) from TBC9-6HA and RHΔku80Δhxgprt lines were collected and lysed on ice for 40 min using Native lysis Buffer (Solarbio R0030) supplemented with PMSF protease inhibitor. The lysates were centrifuged at 21, 130 g for 15 min at 4 °C, and input samples were collected. The supernatants were then incubated with Anti-HA Magnetic Beads (BeyoMag™ P2121) overnight at 4 °C on a rotating shaker. After collecting the unbound samples, the magnetic beads were washed three times with 500 µl of 1XTBS. The magnetic beads were resuspended in PBS with 5xLaemmli sample buffer for the detection of bound samples. The purified samples were subjected to SDS-PAGE and silver staining prior to mass-spectrometry analyses. For Western blots, collected parasites were resuspended in PBS and mixed with 5xLaemmli sample buffer. Protein samples were heated at 95 °C for 10 min and were separated on 10% acrylamide gels, followed by blotting using a BioRad wet-blotting system. Proteins were detected using specific primary antibodies and secondary antibodies conjugated with LI-COR 800CW or 680CW reagents. The membranes were visualized using a Bio-Rad ChemiDOC MP imaging system.

For Western blotting experiments, cells were lysed for 30 min at 4°C in HEPES‐phospho‐lysis buffer and the protein concentrations determined using the Pierce™ BCA assay (Thermo Scientific). Then, the same amounts of protein lysates were loaded onto NuPAGE™ Novex™ 4–12% Bis‐Tris precast polyacrylamide gels (ThermoFisher) according to the manufacturer's instructions. Following, proteins were transferred onto a nitrocellulose membrane (GE, 10401197) using a BioRad^® wet blotting system. The membrane was blocked in 3%‐milk PBS‐T (phosphate‐buffered saline, 0.05% Tween) for 60 min at room temperature and incubated with primary antibody in 3%‐milk PBS‐T overnight at 4°C. Bound antibodies were detected with an appropriate HRP (horse‐radish‐peroxidase)‐coupled secondary anti‐rabbit (1:2,000, Sigma A0545) and anti‐mouse (1:2,000, Sigma A0168) antibody by measuring chemiluminescence in a Fujifilm LAS‐3000 after the addition of WesternBright Quantum (Advansta, 12042‐D20). The primary antibodies used were as follows: anti‐NanoLuc (rabbit, 1:5,000, kindly provided by Promega), anti‐GFP (rabbit, 1:5,000, abcam ab290), anti‐CSP (rabbit, 1:1,000, Synaptic Systems, 154 003), anti‐Histone H3 (rabbit, 1:5,000, abcam ab1791), anti‐VCP (mouse, 1:1,000, Progen, 65278), anti‐UBXD9 (rabbit, 1:1,000, LifeSpan Biosciences, LS‐C156546, EPR8616), and anti‐tubulin (mouse, 1:4,000, Sigma, T6074).

Parasites were resuspended in PBS, followed by addition of 5× Laemmli sample buffer supplemented with 1 mM D-Dithiothreitol (DTT) for a further incubation at 98°C for 10 min. Protein lysis was then resolved in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), blotted by a Bio-Rad wet-blotting system, subsequently incubated with different combinations of primary and secondary antibodies, followed by secondary antibodies conjugated with LI-COR reagents, or streptavidin LI-COR 800CW. The blot membranes were then visualized using a Bio-Rad ChemiDOC MP system.