Anti c myc agarose beads

A volume of ~20 µL of either rabbit anti–c-Myc agarose beads (Sigma-Aldrich), mouse-HA agarose beads (Sigma-Aldrich), or protein G agarose beads (Sigma-Aldrich) were equilibrated through three separate washes with 1% Triton X-100 in PBS + PI. The protein G beads were then conjugated to 20 µL of rabbit EXP2 antibody by incubating both together for a minimum of 1 h at 4°C with constant agitation. Parasites extracted with 0.1% saponin (Sigma-Aldrich) were lysed with 1% Triton X-100/PBS + PIs and following incubation for 1 h at 4°C, the lysate was centrifuged at 10,000 × g for 5 min at 4°C. The supernatant was then incubated with unconjugated protein G agarose beads for a minimum of 1 h at 4°C with constant agitation to pre-clear lysate of unspecific protein G binding. The lysate was then incubated with the equilibrated anti–c-Myc agarose or EXP2-conjugated protein G beads for 4 h at 4°C with constant agitation, and the beads were washed three times with 1% Triton X-100/PBS + PI followed by two washes in 1× PBS + PI. For Western blotting, precipitated proteins were eluted off the beads by incubating with pre-heated (60°C) 2× non-reducing sample buffer [1% sodium dodecyl sulfate (SDS), 10% glycerol, 1 mM EDTA, 0.005% bromophenol blue, and 50 mM Tris hydrochloride (pH 6.8)] for 5 min. Dithiothreitol was added to the eluted proteins at a final concentration of 50 mM.

The Co-IP procedure was performed as described previously 28 (link). Briefly, different combinations of plasmids were cotransfected into cells, followed by incubation at 37°C for 48 h. The transfected cells were then lysed in 1×RIPA lysis buffer supplemented with a protease inhibitor cocktail. After centrifuging at 13,000 rpm for 15 min, the supernatant was incubated with anti-Flag agarose beads and anti-c-Myc agarose beads (Sigma-Aldrich, #A7470) at 4°C for 2 h. The beads were then washed 5 times with 1×RIPA lysis buffer, and protein interactions were determined using immunoblot assays.

Immunoprecipitation of MUC17-3TR was performed on ice. Magnetic Protein G Dynabeads (Life Technologies) were washed in PBS and coated with anti-MUC17C1 antibody in PBS for 5 h at 4°C. Unbound antibody was removed by washing 5× with 1% (v/v) Igepal CA-630 (Sigma) in PBS. Alternatively, pre-coated anti-c-Myc Agarose Beads (Sigma, A7470) were used. Cells were harvested by scraping and lysed in 50 mM Tris pH 7.5, 1 M NaCl, 10 mM MgCl₂, cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche), 5% (v/v) glycerol, 1% (v/v) Triton-X 100, 1 mM EDTA and 1 : 100 phosphatase inhibitor cocktails 2 and 3 (Sigma). Debris was removed by centrifugation at 20 000×g and 4°C for 10 min and supernatant bound to anti-MUC17C1 coated protein G Dynabeads or anti-c-Myc Agarose Beads at 4°C overnight. Unbound material was removed by washing in 1% (v/v) Igepal CA-630 in PBS.

For immunoprecipitation 50 ul of Anti-c-Myc agarose beads (Sigma) were washed before adding the samples with Phosphate-Buffered Saline (PBS) 1x. Samples (about 1 g) of mock-, HA-WMV-P1, MYC-CYSDV-P25, or HA-WMV-P1 + MYC-CYSDV-P25 agroinfiltrated N. benthamiana leaves were collected, ground in immunoprecipitation buffer (20 mM Tris-HCl pH 7.5, 30 mM NaCl, 1 mM EDTA, 0,5% NP-40, 2% b-mercaptoethanol) and cleared by centrifugation at 13,200 rpm for 10 min at 4°C. Supernatants of the different lysates were added to samples of washed beads and incubated for 1 h at 4°C. After immunoprecipitation, beads were washed three times with ice-cold PBS 1x for 1 min each. Input extracts and eluates of immunoprecipitations were used for Western blot analysis (see above).

About 2.5 x 10⁶ logarithmically growing NIH/3T3 cells, stably transfected with pBSwitch and inducible pSLHGC-MYC-AUF1 p42 [4 (link)], were induced for myc-AUF1 expression with 1 nM mifepristone for 24 h. Cells were lysed in 750 μl CelLytic-M lysis reagent (C-2978; Sigma, St. Louis, MO), cleared by centrifugation at 10,000xg and the supernatant incubated at 4°C for 4 h with anti-c-myc agarose beads (A-7470; Sigma) according to the manufacturer’s protocol. Immunoprecipitates were harvested by 15 sec centrifugation at 10,000xg and the beads washed twice with 1 x IP buffer (I-5779; Sigma), and resuspended in 100 μl of 0.1 x IP buffer. RNA was isolated using Qiagen RNeasy minikit (Qiagen, Hilden, Germany) after DNAse I digestion according to the Qiagen manual. Control cells were not induced with mifepristone but the rest of the procedure was kept identical. A small fraction of mRNA was quantified on a NanoDrop apparatus (ND-1000, NanoDrop Technologies, Wilmington, DE) and the RNA quality analyzed using an Agilent Bioanalyzer chip (Agilent Technologies, Santa Clara, CA). All samples showed a similar pattern with a clear 18S rRNA peak, submolar amounts of 28S rRNA, some specific degradation products of rRNA, and mRNA. RNP-IPs of control cells differed only in their threefold lower RNA concentration.

HEK293T cells transfected with the indicated plasmids were harvested 36–48 h post-transfection. Cells were lysed with NETN150 lysis buffer and incubated with pre-equilibrated anti-FLAG M2 affinity gels (Sigma-Aldrich), anti-HA magnetic beads (Thermo Fisher), or anti-c-Myc agarose beads (Sigma-Aldrich) for 4 h at 4 °C with gentle rocking. After incubation, the beads were washed 3–4 times in NETN150 lysis buffer, and protein complexes were eluted by boiling in 2× Laemmli sample buffer for 5 min. For subcellular fractionations, cells were collected by trypsinization, washed with ice-cold PBS, lysed in cytoskeleton (CSK) buffer (100 mM NaCl, 10 mM Tris-HCl pH 6.8, 300 mM sucrose, 3 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100) complemented with protease and phosphatase inhibitor cocktails and incubated on ice for 5 min. Lysates were pelleted at 1500 × g for 5 min, and the supernatant, labeled as S, was collected and the concentration measured. Pellets, designated as P, were resuspended with a 1:1 ratio of PBS and 2× boiling buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 850 mM β-mercaptoethanol) and boiled for 15 min.

For IP analyses, the pcDNA3-2×Flag-CtBP1 and pcDNA3-2×Flag-CtBP2 plasmids were transfected into HC-OA cells. After incubation for 48 h, cells were lysed with 1×RIPA buffer supplemented with a protease inhibitor cocktail (Roche, Shanghai, China, #11697498001). Cell lysates were centrifuged at 14000 rpm for 15 min, followed by incubation with anti-Flag-agarose (Sigma-Aldrich, #A4596) at 4°C for 2 h. The Flag beads were rinsed with 1×RIPA buffer four times. The resulting Flag-CtBP-associated protein complexes were subjected to immunoblot analyses. For Co-IP analysis, the procedures were the same as a previous protocol 35 (link). Briefly, different combinations of Flag-tagged and Myc-tagged plasmids as shown in figures were co-transfected into cells, respectively. After incubation at 37°C for another 48 h, cells were lysed and incubated with anti-Flag agarose beads and anti-c-Myc agarose beads (Sigma-Aldrich, #A7470) at 4°C for 2 h. The beads were rinsed with 1×RIPA buffer four times, and protein levels were examined using western blotting assays.