For 3×FLAG-tag affinity isolation experiments we used HEK293 Flp-In TREx cell lines expressing the tagged protein of interest, established according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Fusion proteins were introduced using a modified pcDNA5/FRT/TO vector that contains the protein of interest followed by a C′-terminal 3×FLAG-tag (Supplementary Figure S1). For GFP-tag affinity isolation, HeLa Kyoto cell lines expressing LAP-tagged proteins of interested were provided by Poser et al. (14); the LAP-tag includes enhanced GFP (EGFP) (13).Protein expression
For 3×FLAG-tagged proteins, expression was induced following the manufacturer's instructions (Invitrogen) by replacing cell growth media (DMEM, high glucose, GlutaMAX, no. 61965; Invitrogen), supplemented with 10% FBS (dialyzed, U.S., no. 26400; Invitrogen) and 1:100 v/v penicillin-streptomycin liquid (no.15140; Invitrogen), with fresh media containing tetracycline (no. 87128; Sigma-Aldrich, St. Louis, MO, USA). Expression levels of the endogenous and 3×FLAG-tagged RRP6 protein were monitored by Western blotting analysis using antibodies to the endogenous protein (no. ab95028; Abcam, Cambridge, MA, USA) and an anti-rabbit, HRP-conjugated secondary antibody (no. P0160; Dako, Glostrup, Denmark). Tetracycline quantities used during protein induction were titrated accordingly, to yield near wild-type (WT) expression levels for the tagged-protein. For LAP-tagged proteins, HeLa cells were grown in the same media as HEK cells, but additionally supplemented with 400 µg/mL G418 (no. 11811; Invitrogen). LAP-tagged proteins are expressed under control from the WT promoter and gene context (14, 36, 37).Cell lysis methods
Cells were lysed by either sonication or cryogenic grinding. Briefly, freshly grown cells were scraped from one 150-cm2 culture dish (Dishes Nunclon Δ, no. 168381; Nalge Nunc, Rochester, NY, USA) in extraction buffer and were sonicated: 20 W, 3 cycles of 10 s each, using a microtip probe and cooling on ice 10 s in between each cycle. For cryogenic disruption, cells were ground using a Retsch (Haan, Germany) PM 100 planetary ball mill by the following program: ~1 g WCW of frozen cells were placed in a 50-mL grinding jar with 3 × 20 mm stainless steel balls, all pre-cooled with liquid N2, and run for three cycles of 3 min, 400 rpm, cooling with N2 in between each 3 min cycle. The 20-mm balls were then replaced with 18 × 10 mm pre-cooled stainless steel balls. The cell powder was subjected to an additional six cycles of 1 min each, 400 rpm, with N2 cooling in between. The resulting cell powder is stored at -80°C. A detailed protocol is provided in supplement. In both cases >90% cell disruption was confirmed by light microscopy.Antibody coupling
All antibodies were coupled to Dynabeads M-270 epoxy (no. 143-02D; Invitrogen). The optimal concentration for coupling to anti-FLAG antibodies (nos. F3165 and F1804; Sigma-Aldrich) was determined as described in the text. In all subsequent cases, anti-FLAG and anti-GFP antibodies were coupled at a concentration of 10 μg antibody/mg of Dynabeads (22, 38). After antibody coupling and final washing, 200 μL storage buffer were added to 30 mg equivalent Dynabeads (dry weight). Dynabeads can be stored at 4°C in PBS supplemented with 0.02% w/v sodium azide or PBS in 50% v/v glycerol for storage at -20°C. Polyclonal anti-GFP antibodies were generated in llama, otherwise prepared as described (22). See Rothbauer et al. (28) for details of the anti-GFP nanobody.Affinity isolation and SDS-PAGE
Briefly, affinity media were added to clarified cell extracts, centrifuged at 14,000 rpm, 10 min in a microfuge, and incubated with rotation at 4°C for 1 h. Affinity media were prewashed with 3 × 1 mL extraction buffer and again with 3 × 1 mL extraction buffer after the 1 h incubation. For elution of the exosome and NEXT complexes, 1 × LDS sample buffer (no. NP0007, no reducing agent; Invitrogen) was added directly to the affinity media, with incubation at 75°C for 10 min, and the supernatant collected. A detailed protocol is provided in the supplementary material. For elution of the CBC complex, 1 mg/mL 3×FLAG peptide (no. F4779; Sigma-Aldrich) was added to the affinity media and incubated 15 min at room temperature. Electrophoresis was performed using NuPAGE 4%–12% Bis-Tris gels (Invitrogen) as per manufacturer's instructions or as specified in text. Bands were visualized by Coomassie staining (39). Bands of interest were excised and analyzed using a MALDI-prOTOF mass spectrometer (PerkinElmer, Waltham, MA, USA) and mass-to-charge ratio (M over Z) (40), PeakErazor (41), and ProFound (42) (http://prowl.rockefeller.edu/prowl-cgi/profound.exe) software. See the supplementary materials for sample work-up details, raw data, and a full results summary; additional details contained in the text.