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. 2018 Mar 14;27(10):1794–1808. doi: 10.1093/hmg/ddy095

Figure 3.

Figure 3.

TMEM127 interacts with and affects ragulator complex abundance. (A) Ctrl and two independent clones of TMEM127 KO HEK293FT cells generated by CRISPR-Cas9 (KO1, KO2) were transduced with lentivirus carrying either pLKO EV or pLKO-shLAMTOR1 (shL1) construct. Cells were stimulated with amino acids for 15 min after a 2-h amino acid starvation, as described in Figure 1, 72 h after transduction. Blots were probed with antibodies for phosphorylated S6 kinase (p‐S6K T389) and S6 (p‐S6 S240/244), their corresponding total protein (T‐S6K and T‐S6, respectively), LAMTOR1, TMEM127 and β‐actin (loading); three biological replicates were performed. (B) Flag IP of HEK293T cells expressing Flag‐LAMTOR1 or Flag-EV and probed for endogenous TMEM127. Corresponding WCLs were probed with Flag, TMEM127 and β-actin; three biological replicates were performed. (C) HA IP of HEK293T cells expressing HA-TMEM127 or HA-EV and probed for endogenous LAMTOR1. Corresponding WCLs were probed with HA, LAMTOR1 and β-actin antibodies; three biological replicates were performed. (D) Endogenous TMEM127 IP in HEK293T cells using a polyclonal TMEM127 antibody and probed for endogenous LAMTOR1; IgG was used as a negative control, corresponding WCLs are shown. (E) Flag IP of HEK293T cells expressing Flag-TMEM127 and HA-LAMTOR1 WT or HA-LAMTOR1 G2A mutant and probed for HA and Flag; corresponding WCLs are shown and β-actin controls for loading; three biological replicates were performed. (F) Flag IP of HEK293T cells expressing Flag-TMEM127 and probed for endogenous LAMP1 and TMEM127 (both Flag-TMEM127 and endogenous TMEM127 bands are seen on WCLs, shown on the left, β-actin controls for loading. (G) Confocal microscopy of GFP‐TMEM127 in HEK293T control (LAMTOR1 WT) and LAMTOR1 KO cells; scar bar: 10 μm. (H) Fractionated lysates from Ctrl, two independent clones of TMEM127 KO (1 and 2) and LAMTOR1 KO HEK293FT cells showing membrane fraction containing lysosomes (Lyso), cytosolic fractions (Cyto) and WCLs, probed for TMEM127, LAMTOR1, LAMTOR2 and LAMTOR4, and RagC. Tubulin and LAMP2 were used as cytosolic and lysosomal markers, respectively; three biological replicates were performed. (I) WCLs of WT and Tmem127 KO MEFs and probed for endogenous LAMTOR1, LAMTOR2, TMEM127, β-actin controls for loading; three biological replicates were performed. (J) Western blot of HEK293T cell lysates expressing increasing amounts of GFP‐TMEM127 (0–500 ng) and probed for endogenous LAMTOR1, LAMTOR4, RagC, LAMP1 and TMEM127, β‐actin is a loading control; three biological replicates were performed (quantification shown in Supplementary Material, Fig. S3G). (K) WCLs of three pheochromocytomas carrying distinct truncating TMEM127 mutations (MUT samples #2, 3, 4) and four pheochromocytomas with WT TMEM127 sequence (WT samples #1, 5, 6, 7) probed with TMEM127, LAMTOR1, LAMTOR2, RagA, RagB, ATP6v0d1 and β-actin, as loading control. Note the absence of detectable WT or truncated TMEM127 protein in mutant lanes; one of two western blots run with the same panel of samples is shown. (L) Flag IP of HEK293T cells expressing Flag-LAMTOR1 and transfected with the indicated amount of HA-TMEM127-WT or HA-TMEM127 532DupT mutant construct, probed for HA and Flag; corresponding WCLs are shown on the left and β-actin controls for loading. Three biological replicates were performed.