To identify G55- and G45-interacting proteins, H1299 cells were transfected with GFP-G55 or HA-G45, lysed in 1× RIPA buffer, and subjected to IP with anti-GFP or anti-HA antibodies, respectively, linked to agarose G beads. Immune complexes were subjected to LC-MS. To identify G55-dependent secreted proteins, CM samples were collected, concentrated, and subjected to LC-MS as previously described (49). Samples were solubilized with 5% SDS, 50 mM TEAB, pH 7.55, final volume 25 μL. The sample was then centrifuged at 17,000g for 10 minutes to remove any debris. Proteins were reduced by making the solution 20 mM TCEP (Thermo Fisher Scientific, 77720) and incubated at 65°C for 30 minutes. The sample was cooled to room temperature and 1 μL of 0.5 M iodoacetamide acid added and allowed to react for 20 minutes in the dark. Next, 2.75 μL of 12% phosphoric acid was added to the protein solution, and 165 μL of binding buffer (90% methanol, 100 mM TEAB, final pH 7.1) was then added to the solution. The resulting solution was added to S-Trap spin column (protifi.com) and passed through the column using a benchtop centrifuge (30-second spin at 4,000g). The spin column was washed with 400 μL of binding buffer and centrifuged. This was repeated 2 more times. Trypsin was added to the protein mixture in a ratio of 1:25 in 50 mM TEAB, pH 8, and incubated at 37°C for 4 hours. Peptides were eluted with 80 μL of 50 mM TEAB, followed by 80 μL of 0.2% formic acid, and finally 80 μL of 50% acetonitrile, 0.2% formic acid. The combined peptide solution was then dried in a speed vac and resuspended in 2% acetonitrile, 0.1% formic acid, 97.9% water and placed in an autosampler vial.

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