Fig. 4: Lamtor1 interacts with MPRIP independently of mTORC1.

a Interacting of Lamtor1 and MPRIP. FLAG-tagged Lamtor1 expression vector or control vector was transfected into V5-tagged-MPRIP-expressing HEK293T cells. Cells were lysed, precipitated with anti-V5 antibody (left) or anti-FLAG antibody (right), and detected by western blotting with anti-V5 and -FLAG antibodies. Whole-cell lysates (WCL) were detected with anti-β-actin antibody. Data are representative of three experiments. b Co-localization of Lamtor1 and MPRIP. Localization of MPRIP in Lamtor1-FLAG-expressing THP1 was evaluated by confocal microscopy using anti-MPRIP and anti-FLAG antibodies. A representative image of MPRIP (green) and Lamtor1 (red) in a polarized cell is shown. Scale bar, 10 μm (upper), and the intensity of MPRIP (green) and Lamtor1 (red) signals (bottom) (left). Percentage of co-localization of Lamtor1 and MPRIP in the body or tail region of polarized cells (n = 40) (right). c Lamtor1 interacts with MPRIP independent of mTORC1. Forty-eight hours after transfection of FLAG-Lamtor1 expression vector or control vector into V5-MPRIP-expressing HEK293T cells, the cells were treated with 10 nM rapamycin for 2 h. The lysates were precipitated with anti-V5 (left) or anti-FLAG antibody (right), and the immunoprecipitates were subjected to western blotting with anti-V5 and -FLAG antibodies. WCL was blotted with anti-β-actin, anti-S6K, and anti-phospho-S6K antibodies. Data are representative of three experiments. Statistical analyses were performed by two-sided Mann–Whitney U test (b) [median; 25th and 75th percentiles; and minimum and maximum of a population excluding outliers; ***p < 0.0001].