Rfp trap beads

After washing with PBS, cell lysis was performed using immunoprecipitation (IP) buffer (50 mM Tris pH 8, 120 mM NaCl, 0.5% NP40, 1 mM EDTA). Immunoprecipitation was performed using GFP- or RFP-trap beads (Chromotek). Precipitates were washed in IP buffer. Input and precipitate samples were then processed for SDS-PAGE and Western blotting.

Detailed protocols can be found at 10.17504/protocols.io.8epv5xj9ng1b/v1. Double-knockout (YIPF3^−/−YIPF4^−/−) HEK293 cells were reconstituted with mCherry–YIPF4 (WT or LIR mutant) and GFP–YIPF3 (WT or LIR mutant) constructs and sorted for equal expression levels. Immunofluorescence was used to confirm proper localization of both YIPF3 and YIPF4. Then cells were plated on 10-cm plates and grown to 70% confluency. Cells were left untreated or starved using amino acid withdrawal for 2 h in the presence of BafA1 (100 nM). Cells were washed twice with cold PBS and then lysed in 0.8 ml NP-40 lysis buffer (100 mM Tris pH 7.4, 150 mM KCl, 0.1% NP-40, 0.5 mM EDTA, 1× HALT (Roche) protease inhibitors, PhosSTOP tabs). A 1.5 mg quantity of protein from each sample was added to 15 μl of washed RFP–TRAP beads (ChromoTek, number rta) and incubated for 2 h while rotating at 4 °C. Beads were washed three times with lysis buffer and eluted in 1× LDS loading dye at 94 °C for 5 min. For Flag–LC3B immunoprecipitation, 1.5 mg of protein from each sample was added to 20 μl of washed Pierce anti-Flag beads (number A36797) and incubated for 2 h while rotating at 4 °C. Beads were washed three times with lysis buffer and eluted in 1× LDS loading dye at 94 °C for 5 min.

Cell lysis was performed using immunoprecipitation (IP) buffer (50 mM Tris–HCl, pH 8, 120 mM NaCl, 0.5% NP40, 1 mM EDTA). Lysates were centrifuged at 20,000 × g for 15 min at 4 °C. Immunoprecipitation from supernatants was then performed using 20 μl of RFP-trap beads (Chromotek, Munich, Germany; 2 h at 4 °C on a rotator). Precipitates were washed in IP buffer and then processed together with input samples for SDS-PAGE and Western blotting.

Brains were lysated in extraction buffer [25mM HEPES pH6.8, 50mM KCl, 1mM MgCl2, 1mM DTT, 125mM sucrose, protease inhibitor and RiboLock RNase Inhibitor (Thermo Scientific)]. After 10min centrifugation at 4°C, lysate was applied to RFP-Trap beads (ChromoTek) and incubate for 1h at 4°C on a rotative wheel. Beads were washed five times in RIPA buffer [50mM Tris-HCl pH7.5, 1% NP-40, 1% sodium desoxycholate, 0.1% SDS, 1mM EDTA and 1mM NaCl]. Samples were then processed for western blotting. Experimental conditions were repeated twice to account for technical and biological variation.

GFP- and RFP-tagged transgenes were immunoprecipitated using GFP- and RFP-TRAP beads (Chromotek), respectively, according to the manufacturer’s instructions. V5-tagged transgenes were purified using V5-agarose beads (Sigma). For Ago3 and Piwi5 IP experiments, antibodies targeting endogenous proteins were added to lysates at 1:10 dilution and incubated for 4 h at 4°C, followed by overnight binding to Protein A/G PLUS agarose beads (Santa Cruz). Protein extracts were resolved on polyacrylamide gels, blotted to nitrocellulose membranes, and probed with the indicated antibodies. Details on generation of antibodies, experimental procedures, and antibody dilutions can be found in the Supplementary Data.