Ha magnetic beads

WT or HA-Rab11a expressing A549 cells infected with RSV-GFP-N, RSV-L-GFP, RSV-GFP or RSV-WT for 16 hours were lysed in a co-IP lysis buffer (Tris 25mM pH 7.2; NaCl 150 mM; IGEPAL CA-630 0.2% (Sigma); glycerol 10% (v/v); EDTA 0.5mM; antiprotease and phosphatase (Thermofisher)), then incubated overnight at 4°C with GFP-Trap beads (Chromotek) or Magnetic-HA beads (Thermofisher) according to the manufacturer’s recommendations. Beads were rinsed three times in a co-IP dilution buffer (Tris 25mM pH 7.2; NaCl 150mM), then eluted in Laemmli buffer at 95°C and analyzed by SDS-PAGE.

Lysosome immunoprecipitation was carried out as described^{14 (link)} with a few modifications. All steps of the process were carried out at 4 °C with cold solutions. Briefly, HeLa TMEM192-3xHA endogenously tagged cells that were grown to 80% confluency in 150-mm plates were washed twice with PBS, scraped into tubes, and then pelleted at 500×g for 5 min. The cells were re-suspended in 2 mL of lysoIP buffer (50 mM KCl, 100 mM KH₂PO₄, 100 mM K₂HPO₄, pH 7.2) supplemented with protease and phosphatase inhibitors (Roche). The cells were transferred to a glass homogenizer and dounced using 25 strokes. The lysed cells were centrifuged at 1000×g for 10 min, and the post-nuclear supernatant (PNS) was collected. The concentration of the PNS was determined by Bradford assay. Lysosomes from normalized amounts of PNS were immunoprecipitated by incubation with 50 μL of magnetic HA-beads (Thermo Scientific Cat#88837) for 1 h on a rotator. The beads were sequentially washed once with lysoIP buffer containing 300 mM NaCl and once with lysoIP buffer. The lysosomes were solubilized and eluted off the beads by incubating the beads with 150 μL of lysoIP buffer with 0.5% NP-40 in a thermomixer (1000 rpm) for 30 min. The eluates were snap frozen in liquid nitrogen and stored in −80 °C.