Protein g bead

Co-immunoprecipitation assays were performed to precipitate either protein or RNA. Gametocyte lysates of WT NF54 and the 7-Helix-1-HA line were obtained as described above. For the precipitation of proteins, the lysates were initially incubated with 5% v/v pre-immune mouse or rabbit sera and 20 μl of protein G-beads (Roche) for 1 h at 4°C. After centrifugation, the supernatant was incubated for 1 h at 4°C with 5% v/v polyclonal mouse antisera against 7-Helix-1, Puf2 as well as Pf39 [94 (link)] used as a negative control, or polyclonal rabbit antisera against CITH, DOZI or HA. A volume of 20 μl protein G-beads was added and incubated overnight at 4°C. The beads were centrifuged, washed with PBS five times and resuspended in an equal volume of loading buffer. The sample was then subjected to WB (see below). For the precipitation of RNA (RIP; [53 (link)]), the beads were resuspended in Trizol instead of loading buffer, and RNA isolation and diagnostic RT-PCR were performed as described below.

To immunoprecipitate RISC-RNA complexes we adapted the protocol from Hendrickson et al. [24 (link)]. HeLa cells were cultured in 10 cm² tissue culture plates and transiently transfected with either miRNA mature duplexes or Control RNA duplexes at a final concentration of 100 nM or mock transfected. 24 hours post transfection, cells were washed 2X with PBS, 0.5 mL of lysis buffer was added to the plate, followed by incubation at 4°C for 30 minutes. Cell lysates were collected by scraping and debris cleared by centrifugation at 14,000 rpm at 4°C. 50 μl of the lysate was collected as input for total RNA profiling. Lysates were pre-cleared by incubating with 50 μl of protein-G beads (Roche) for 1 hour at 4°C and then collecting supernatants (pre-clearing). Pre-cleared lysates were then incubated with 15 μg of antibody against Argonaute-2 (AGO2)(ab57113, Abcam) at 4°C for 3 hours. AGO2 is a protein component of RISC. Following AGO2 antibody incubation, 50 μl of protein-G beads were added to the lysate and incubated for 1 hour at 4°C. Beads were washed 8 times with lysis buffer and RISC-RNA complexes were extracted by adding 1 mL TRIzol reagent (Invitrogen) directly to the beads. RNA extraction with TRIzol was carried out as per the manufacturers instructions.

Nine-day-old liquid-cultured seedlings of transgenic plants harboring 35 S::MtSERK1-FLAG, or 35 S::MtSERK1-FLAG and 35 S::MtBRI1-GFP transgenes were treated with or without 24-epiBL for 90 min and ground to fine powder in liquid nitrogen for total protein preparation. MtSERK1-FLAG was immunoprecipitated with agarose-linked α-FLAG antibody (Sigma, St. Louis, MO). For phosphorylation analysis, transgenic plants of 35 S::MtBRI1-GFP were treated with mock solution or 24-epiBL for 90 min, and ground to fine powder in liquid nitrogen for membrane protein isolation^{21 (link)}. MtBRI1-GFP was immunoprecipitated with the α-GFP antibody (Invitrogen, Carlsbad, CA) and protein G beads (Roche, Indianapolis, IN). The immunoprecipitated proteins were separated on 7.5% SDS polyacrylamide gel for western blot analyses with α-GFP, α-FLAG or α-phosphothreonine antibodies as previously described^{60 (link)}.

Kinase assays were performed with 1, 1.5, and 2 μg of 167V VRK2 and 2 μg of 167I VRK2. The VRK2 forms were expressed in Escherichia coli using the pTYB2 vector (intein tag) and purified by pull-down assays using the chitin bead system. The reaction was performed at 37°C in a kinase buffer solution [20 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 0.5 mM dithiothreitol, 150 mM KCl, and (γ-³²P)ATP]. The reaction was stopped after 1 h, and proteins were separated by SDS polyacrylamide gel electrophoresis, and visualized with autoradiography.
The immunoprecipitated VRK2 was obtained from flag-tagged VRK2-overexpressing 293A cells and MDA-MB-231 cells. The harvested cells were lysed in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM EDTA, and 10 mM ethylene glycol tetraacetic acid [EGTA]) and immunoprecipitated with anti-Flag antibody (F1804, Sigma-Aldrich, MO, USA) and protein G beads (05 015 952 001, Roche, Basil, Switzerland). VRK2 with attached G beads was used for the kinase assay. After the kinase reaction, proteins were eluted from the beads by denaturation at 95°C.

HeLa-RIPK3 (5 × 10⁶) and HaCaT were used for each group. Cells were incubated with 2 µg of Fc-Flag-TWEAK^{27 (link)} for 10 or 30 min (at 37 °C and 5% CO₂) or remained untreated as a control. After that, cells were washed four times with ice-cold PBS to stop receptor complex formation. Cells were lysed on ice by mixing with 2 ml of lysis buffer (30 mM Tris-HCl, pH 7.5, 120 mM NaCl, 50 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10% glycerol, 1% Triton X-100, protease inhibitor mixture (Roche Molecular Diagnostics). Lysates were centrifuged twice (4 min, 1200 r.p.m., 4 °C) followed by another centrifugation for 30 min to remove cellular debris. A minor fraction of the resulting clear lysates was used to control for the input of the respective proteins. Lysates of untreated control cells were supplemented with 5 ng Fc-Flag-TWEAK. Receptor complexes were precipitated from the lysates by co-incubation with 40 µl of protein G beads (Roche Applied Science) overnight on a shaker at 4 °C. The beads were washed with 2 ml of lysis buffer four times by centrifugation (30 s, 5000 r.p.m., 4 °C). Finally, the pellets were mixed with lysis buffer and 4 × sample buffer and were heated at 85 °C for 10 min. After removal of the remaining insoluble debris by centrifugation (2 min, 1200 r.p.m.), the indicated proteins were detected by western blotting.