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. Author manuscript; available in PMC: 2019 May 18.
Published in final edited form as: Science. 2018 Apr 26;360(6390):751–758. doi: 10.1126/science.aar2663

Fig. 3. NUFIP1-ZNHIT3 interacts with ribosomes in an mTORC1-dependent fashion.

Fig. 3

(A) mTOR inhibition increases the amount of NUFIP1-ZNHIT3 that co-migrates with ribosomes. HEK-293T cell lysates prepared from cells in full media or treated with 250 nM Torin1 were fractionated over a 50% sucrose cushion. Fractions were collected and the indicated proteins analyzed by immunoblotting.

(B) Amino acid deprivation increases the amount of ribosomes that co-immunoprecipitates with NUFIP1. HEK-293T cells stably expressing FLAG-NUFIP1 were deprived of amino acid for 1 hour. Lysates and FLAG immunoprecipitates were prepared and analyzed for the indicated proteins by immunoblotting. FLAG-Rap2A was used as a negative control.

(C) In vitro, purified NUFIP1-ZNHIT3 binds to ribosomes and the interaction is not affected by whether or not NUFIP1-ZNHIT3 was obtained from cells with inhibited mTOR. The FLAG-NUFIP1-HA-ZNHIT3 complex was purified from HEK-293T cells in full media or treated with 250 nM Torin1 for 1 hour and immobilized on a FLAG affinity resin. Equal amounts of ribosomes obtained from cells in full media were added to the immobilized FLAG-NUFIP1-HA-ZNHIT3 complex and the proteins captured analyzed by immunoblotting. Ribosomes were purified as described in the supplementary materials. Purified HA-METAP was used as a negative control.

(D) In vitro, ribosomes purified from cells with mTOR inhibition bind better to NUFIP1-ZNHIT3 than those from cells in full media. Ribosomes were purified from HEK-293T cells in full media conditions or treated with 250 nM Torin1 for 1 hour. The FLAG-NUFIP1-HA-ZNHIT3 complex was immobilized on FLAG affinity beads and equal amounts of ribosomes were added. Proteins captured by the FLAG affinity beads were analyzed by immunoblotting. HA-METAP2 served as a negative control.