Fig. 4

Arl5b interacts with Ragulator through Lamtor1. HEK293T cells were used. a Arl5b, but not Arl1, specifically pulled down Lamtor1-GFP. Bead-immobilized GST-Arl5b or Arl1 was in vitro loaded with GDP or GMPPNP and subsequently incubated with cell lysates expressing GFP or Lamtor1-GFP. Pull-downs were analyzed by immuno-blotting GFP fusions. The loading of GST fusions were shown by Coomassie blue staining. 1 and 2 indicate Lamtor1-GFP and GFP band, respectively. b, c Arl5b-QL and -TN interact with Lamtor1-Myc in forward and reverse co-IPs. Cells co-expressing indicated tagged-proteins were incubated with indicated antibodies and IPs were immuno-blotted against indicated tags. Lamtor2-GFP and SNX3-Myc served as a positive and negative control, respectively. In b, 1 and 2 indicate Arl5b-(wt, QL or TN)-GFP and Lamtor2-GFP band, respectively. In c, 1–5 indicate IgG heavy chain, Arl5b-(wt, QL or TN)-GFP, Lamtor2-GFP, SNX3-Myc, and Lamtor1-Myc band, respectively. d Full length Lamtor1 is required for Arl5b–Lamtor1 interaction. Bead-immobilized GST-Arl5b-TN was incubated with cell lysates expressing indicated fragments of Lamtor1, and pull-downs were analyzed by immuno-blotting GFP-fusions. * denotes the specific band. e Arl5-QL and -TN interact with Lamtor1 but not Lamtor2–5. Bead-immobilized GST-fusion was incubated with cell lysates expressing indicated Myc-tagged Lamtors and pull-downs were analyzed by immuno-blotting Myc-tagged proteins. Lamtor1-Str.-Myc, Lamtor1-G2A-Strep-Myc. f Arl5b-TN interacts with Ragulator through Lamtor1. Bead-immobilized GST-Arl5b-TN was incubated with cell lysates expressing indicated Lamtors, and pull-downs were analyzed by immuno-blotting indicated tags. 1 and 2 indicate DMyc-Lamtor5 and DMyc-Lamtor4, respectively. g Arl5b-GMPPNP, -GDP, and guanine nucleotide empty form interact with Ragulator. Bead-immobilized GST-Arl5b was first loaded with GMPPNP or GDP or stripped off its bound guanine nucleotide by EDTA treatment. Beads were subsequently incubated with cell lysate, and pull-downs were analyzed by immuno-blotting endogenous Lamtor1–4. h Excess Arl5b-TN inhibits the interaction between Rag GTPases and Lamtor1. Cell lysate co-expressing Lamtor1-GFP, Flag-RagB-T54L, and HA-GST-RagC was IPed using anti-GFP antibody in the presence of 0.2 µM recombinant GST-Arl5b-TN or GST. Pull-downs were subsequently immuno-blotted for indicated tags