Fugene 4k

For transient transfections, wild-type HeLa cells were seeded on six well plates and used the following day. pEGFP-N1 was transfected using Fugene 4K (Promega) at 1.5:1 Fugene:DNA ratio following manufacturer protocol. Cells were allowed to incubate for 24 h before use in immunoprecipitation assays.
Cell lysates from tepsin-GFP, pEGFP-N1 transiently-transfected, or mRFP-GFP-LC3B HeLa cells were prepared as described above. Lysate was incubated with unconjugated agarose (control) resin (Chromotek) for 30 min at 4°C to reduce background binding. Tepsin-GFP lysate and pEGFP-N1 transfected precleared lysate added to GFP-trap resin (Chromotek) at a ratio normalized by GFP transfection efficiency. For mRFP-GFP-LC3B, precleared lysate was divided equally among GFP-trap resin (Chromotek) and fresh control resin for coimmunoprecipitation (IP) experiments. Dilution buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 1 cOmplete Mini EDTA-free Protease Inhibitor Cocktail tablet per 20 ml) was added to bring each sample to an equal volume, then IPs were incubated 1 h at 4°C followed by three washes with dilution buffer. IPs were eluted by adding SDS loading buffer to the washed resin pellet and boiling for 5 min at 95°C.

Cells were seeded on six well plates and used in knockdown assays the following day. AP-4 knockdown was achieved using ON-TARGETplus AP4E1 siRNA (J-021474-05; Dharmacon) and tepsin knockdown was achieved using ON-TARGETplus C17orf56 siRNA (J-015821-17; Dharmacon). Control cells were treated with ON-TARGETplus nontargeting siRNA (D-001810-01; Dharmacon). Transfections of siRNA were carried out with Oligofectamine (Thermo Fisher Scientific) with a final siRNA concentration of 7.5 nM (for each AP-4, tepsin, and control siRNA treatment) in complete culture media and assayed 48 h after transfection. Cells were reseeded 24 h after siRNA treatment to a lower cell density on six well plates (with or without coverslips) for Western blotting and immunofluorescence assays. For rescue experiments, cells were subjected to an additional transfection step. Transfection with siRNA was conducted as described above, but cells were seeded into and maintained throughout the experiment in antibiotic-free media (MEM-alpha with 10% FBS, no G418). Four hours after incubation with siRNA treatment, wild-type and mutant tepsin-myc constructs were transfected using Fugene 4K (Promega) at 1.5:1 Fugene:DNA ratio following manufacturer protocol. Replating and a total time course of 48 h from initial siRNA knockdown were maintained as described above.