Figure 3.

TRIM37 interacts with MTOR and RRAGB proteins and its depletion inhibits MTOR-RRAGB interaction and lysosomal distribution of MTOR. (a) HEK 293T cells were transfected with FLAG-MTOR and TRIM37-GFP constructs. After 24 h, equal amounts of cell lysates were collected for immunoprecipitation (IP) with control IgG (Ctrl) or FLAG antibody, followed by protein detection with GFP and MTOR antibodies. (b and c) HEK 293T cells were transfected with HA-GST-RRAGB and TRIM37-GFP constructs. After 24 h, cells lysates were collected (normal). Alternatively, cells were starved for amino acid for 2 h (AA-) and stimulated with amino acid-containing media for 1 h (AA+). Equal amounts of cell lysates were collected for immuno-precipitation (IP) with control IgG (Ctrl) or HA antibody. GFP and HA-HRP antibodies were used to detect TRIM37-GFP and HA-GST-RRAGB, respectively. (d and e) HepG2 control and TRIM37 KD cells were incubated in amino acid-depleted media [38] for 2 h (AA-) and then stimulated with amino acid-containing media (AA+) for 15 min. Nuclei were stained with DAPI. (d) Cells were immuno-stained with MTOR antibody. (e) Cells stimulated with AA (15 min) were co-stained with MTOR and LAMP2 antibodies. The images were acquired by confocal microscopy. Scale bars: 10 μm. (F) HEK 293T control and TRIM37 KD cells were transfected with FLAG-MTOR and HA-GST-RRAGB constructs. After 24 h, equal amounts of cell lysates were collected for immuno-precipitation (IP) with control IgG (Ctrl) or HA antibody, followed by protein detection with the indicated antibodies. s.e. short exposure; l.e. long exposure.