Myc trap

Transfected cells were lysed in buffer A (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.5% NP-40 supplemented with Complete Mini EDTA-free Protease inhibitor cocktail tablets (Roche)) and left to incubate for 20 min on ice. The lysates were cleared by centrifugation (13000 g, 30 min) and the proteins were captured with GFP-trap (Chromotek), Myc-trap (Chromotek) or HA-agarose beads (Sigma) by tumbling end-over-end overnight at 4°C. The beads were washed three times by centrifugation (1000 g, 10 s) in buffer A. Finally, the beads were resuspended in SDS sample buffer (94 mM Tris/HCl pH 6.8, 3% SDS, 19% glycerol and 0.75% β-mercaptoethanol) and analyzed by SDS-PAGE and western blotting.

COS7 cells transfected with plasmids were lysed in IP lysis buffer (1% Triton X-100, 20 mM Tris pH 8, 130 mM NaCl, 1 mM DTT, supplemented with protease inhibitor cocktail complete (Roche) and PhosphoStop (Roche)). After clarifying the samples, lysates were incubated with myc-trap or GFP-trap beads (ChromoTek). The immunoprecipitates were washed with IP lysis buffer, and the proteins were eluted with NuPAGE LDS Sample Buffer (Invitrogen) containing 5% β-mercaptoethanol. Samples were used in other assays or analyzed by immunoblotting. Lysates were resolved on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked using 5% dried milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) and incubated with primary antibody overnight at 4°C. Membranes were washed three times with TBS-T and incubated for 1 h at room temperature with appropriated HRP-labelled secondary antibody. Enhanced chemiluminescence (ECL; GE Healthcare) was used as a detection reagent.

Plant tissues were ground with liquid nitrogen and then resuspended with two volumes (2 ml per gram tissue) of extraction buffer (20 mM HEPES, pH 7.5, 40 mM KCl, 1 mM EDTA, 1% Triton X-100, 0.2 mM PMSF, and 1× protease inhibitor cocktail). The protein mixtures were centrifuged at 4,000 rpm for 5 min. Then, the resulting supernatant was centrifuged at 20,000×g for 15 min. The supernatants were incubated with Streptavidin-agarose (S1638, Sigma, Saint Louis, MO) or an anti-Myc nanobody coupled to agarose (Myc-Trap, Chromotek, Hauppauge, NY) for 1 h at 4°C. Then, beads were washed with an extraction buffer containing 0.1% Triton X-100 and eluted with 2× SDS sample buffer (24 mM Tris-HCl, pH 6.8, 10% glycerol, 0.8% SDS, 2% 2-mercaptoethanol) containing 0.4 M urea. YFP-TbID and BZR1-MH were detected by monoclonal anti-GFP (1:2000, HT801, Transgen Biotech, Beijing, China) and monoclonal anti-Myc antibodies (1:2000, 9B11, Cell Signaling Technology, Danvers, MA), respectively. Biotinylated proteins were detected with streptavidin-HRP (1:2000, 21124, Thermo Scientific, Rockford, IL).

For IP experiments in S2R+ cultured cells, protocol was followed as described [60 (link)] with minor changes: 2 mg of the protein lysates was incubated for 2 h with 10 μl of either Myc-Trap or GFP-Trap beads (Chromotek). To determine the dependence of interactions on RNA, 50 U of RNaseT1 (ThermoFisher) were added to the respective IP. To ensure the activity of RNase T1, lysates were incubated 10 min at RT prior to the incubation of lysate with antibody.
For IP experiments in ovaries, 150 μl of wet ovaries from 3–5 day old flies expressing Venus-Mkrn1 was homogenized on ice in 2 ml of cold IP buffer (1 X PBS, 0.4% Triton X-100, 1 mM MgCl₂, 5% glycerol), containing protease inhibitors and PMSF. The extracts were diluted to 1.5 mg protein/ml. Each extract (0.66 ml) was mixed with 24 μg of anti-pAbp Fab antibody (Smibert lab, [61 (link)]), 17 μg of α-eIF4G rabbit antibody, or 15 μl of rabbit anti-α−Tubulin antibody (Abcam). When present, 100 μg RNase A (Qiagen) was added to the samples. Samples were incubated with rotation at 4°C overnight, then mixed with 30 μl of protein A agarose beads (wet volume, Invitrogen) and incubated with rotation at RT for 1.5 h. The beads were washed three times with IP buffer. Bound material on the beads was eluted by boiling for 2 min in 40 μl of SDS loading buffer. 20 μl of the eluted sample, together with input samples, was used for western blot.

Cells (600 OD₆₀₀ units) were harvested and lysed using a EmulsiFlex-C3 (Avestin, Ottowa, Ontario, Canada) in 20 mL ice cold lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM PMSF, 2 mM EDTA, 2 mM MgCl₂, 1 × Protease inhibitor complete, 5% glycerol). The cell-free protein lysates (centrifugation at 27,000 × g for 30 minutes at 4 °C) were subjected to IMAC purification using Macherey-Nagel™ Protino™ Ni-IDA (Thermo Fisher Scientific, Waltham, Massachusetts, USA) followed by immunoprecipitation with Myc-Trap® (Chromotek). Eluted proteins were separated by SDS-PAGE gels and stained with Coomassie Brilliant Blue.

ChIP was performed as previously described^{38 (link)} with minor modifications, as follows: 100 ml of a 0.5 OD₆₀₀ cell suspension was crosslinked with 1% formaldehyde (Roth) and quenched with 125 mM glycine (Sigma). Following lysis and sonication, solubilized chromatin corresponding to approximately 5–6 × 10⁸, 4 × 10⁸, and 9 × 10⁸ cells was immunoprecipitated with antibodies against Pol II-S5P (25 μl supernatant, kindly provided by A. Ladurner), H3K9me2 (2 μl), and Myc-trap (10 μl, Chromotek), respectively.
RT–qPCR experiments were performed as described above, using the primers listed in Supplementary Table 4. The IP values were divided by the input and corrected for variation by normalizing to the mean of three euchromatin loci (act1⁺, tef3⁺, ade2⁺). The data were shown as relative to the untagged strain.