*2.9. Transfection of Nucleic Acids*

Plasmid DNA was transfected into cells using Lipofectamine 3000 (ThermoFisher) according to the manufacturer's instructions. A549 cells seeded into 24-well plates on coverslips for immunofluorescence assays were transfected with 1 μg/well DNA, and HEK-293T cells seeded into 6-well plates for co-immunoprecipitation (co-IP) assays were transfected with 5 μg/well total DNA (when co-transfected, this amount was split equally between plasmids; i.e., 2.5 μg/well each). The ratio DNA (μg)/lipofectamine 3000 (μL)/P3000 reagen<sup>t</sup> (μL)/Opti-MEM (μL) was 1:3:2:50. Cell media was freshly changed prior to transfection. Plasmid DNA and P3000 were added to half the volume Opti-MEM (ThermoFisher) and mixed by inversion; lipofectamine 3000 was added to the other half of Opti-MEM and mixed. The two solutions were combined, mixed by inversion, and incubated 15 min at RT prior to drop-wise addition to cells.

#### *2.10. Co-IP of Recombinant FLAG- and HA-tagged Proteins*

HEK-293T cells seeded in 6-well plates and used for experiments were washed 1x with PBS and then lysed in 250 μL/well co-IP lysis bu ffer + inhibitors (50 mM Tris HCl pH 7.5, 250 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.02% (w/v) sodium azide, 1% (v/v) NP40, 1:100 phosphatase inhibitor cocktail (VWR), 1:100 protease inhibitor cocktail (Sigma), 250 nM okadaic acid) on ice. Lysates were scraped into new microcentrifuge tubes and incubated on ice for at least 30 min. Lysates were cleared of cellular debris via centrifugation at 14,000 rpm, 15 min, 4 ◦C, with supernatant transferred to fresh tube on ice and quantified as above. For each co-IP, 250 μg of each sample (in a 200 μL volume—excess lysis bu ffer was used to make volume) was incubated overnight at 4 ◦C in tubes on rotator with the following conditions. *For anti-HA*: 10 μL of thoroughly vortexed anti-HA magnetic beads (Cell Signaling Technology, Danvers, MA, USA) were added to each 200 μL sample, and were rotated overnight at 4◦C. *For anti-FLAG*: Pre-binding of 1 μL anti-FLAG antibody (Sigma) to each 200 μL sample was performed on rotator at 4 ◦C for 15 min. Subsequently, 10 μL of vortexed protein G magnetic beads (ThermoFisher) were added to each 201 μL sample, and were rotated overnight at 4 ◦C.

The following day samples were immunoprecipitated using a magnet. Supernatant was discarded and 500 μL co-IP lysis bu ffer + inhibitors was added to each tube. These were briefly inverted to mix and rotated at 4 ◦C for 5 min. Magnetic precipitation and washing was repeated two more times (three washes total) using 300 μL for repeat washes.

Co-IP lysis bu ffer + inhibitors were used to dilute 4X Laemmli sample bu ffer (Bio-Rad) + β-mercaptoethanol to form a 1X sample bu ffer. A total of 50 μL of this bu ffer was added per sample as a final resuspension/elution. Samples were stored at −80 ◦C.

When loading gels, samples were incubated 3 min at 95 ◦C and then placed immediately on ice. Samples were spot centrifuged >10 s to pellet beads. Samples were then analyzed by Western blot as appropriate, with 10–15 μL co-immunoprecipitate loaded per lane.
