Structure of RINL.

<p>(A) Diagram of the structural features of RIN family members. The lower numbers represent the amino acid residues. (B) FLAG-RIN1, RIN2, RIN3, and RINL were transiently co-transfected with myc-amphiphysin II (amph II) into HEK293T cells. Cells lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-myc and anti-FLAG antibodies. Total lysates were immunoblotted with anti-myc antibody. (C) Cell lysates from HEK293T cells were applied to a Superdex 200 Prep Grade gel filtration column. The elution position was compared with those of the globular size markers (upper panel). The fractions (0.5 ml) eluted from the column and total lysate (tot.) were analyzed by SDS-PAGE, and proteins were immunoblotted with anti-RINL antibody.</p

GEF activity of RINL for Rab5 subfamily proteins.

<p>(A–D) HEK293T cells expressing myc-Rab5b (A), Rab21 (B), Rab22 (C), or Rab31 (D) and FLAG-mock, RINL, RIN3, or Rabex-5 were metabolically radiolabeled with ³²P_i for 4 hours. Myc-Rab5 subfamily proteins were immunoprecipitated with an anti-myc monoclonal antibody, and nucleotides associating with each Rab protein were separated by thin-layer chromatography. The radioactivity of GTP and GDP was quantified, and the percentages (%) of each GTP-bound Rab are shown. Total lysates (bottom) and immunoprecipitated samples (middle) from the radiolabeled cells were separated by SDS-PAGE and immunoblotted with anti-FLAG and anti-myc antibodies, respectively. *p<0.05 vs. mock-transfected cells. (E) Myc-Rab3a, 7a, or 11a was co-transfected with FLAG-mock or RINL into HEK293T cells. The percentages of each GTP-bound Rab member in the metabolically radiolabeled cells are shown as described in (A). (F) Myc-Rab5b was co-transfected with wild type (WT), or the DP_AA or YT_AA mutant of FLAG-RINL into HEK293T cells. The percentages of GTP-Rab5b in the metabolically radiolabeled cells are shown as described in (A). Total lysates (bottom) and immunoprecipitated samples (middle) from the radiolabeled cells were separated by SDS-PAGE and immunoblotted with anti-FLAG and anti-myc antibodies, respectively. All data were obtained from more than three independent experiments and are shown as the mean ± S.E. (error bars). **p<0.01 vs. mock-transfected cells.</p

Identification of odin/Anks1a as an interacting molecule with RINL.

<p>(A) HeLa cell lysates were immunoprecipitated with normal rat IgG or anti-odin antibody, followed by immunoblotting with antibodies as indicated. (B) FLAG-RIN family or FLAG-mock were transfected into HEK293T cells. Cells lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with antibodies as indicated. (C) FLAG-RINL and the indicated deletion mutants of myc-odin were transiently transfected into HEK293T cells. Cells lysates were immunoprecipitated with anti-myc antibody, followed by immunoblotting with antibodies as shown. (D) The indicated deletion mutants of FLAG-RINL were transiently transfected into HEK293T cells. Cells lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with antibodies as indicated. (E) Myc-odin and V5-RINL were co-transfected with FLAG-tagged constitutively active (CA, lanes 2 and 4) or mock (lanes 1 and 3) into HEK293T cells. Cell lysates were immunoprecipitated with anti-myc antibody, followed by immunoblotting with antibodies as indicated. Aliquots of total lysates were also immunoblotted with antibodies as indicated.</p

RINL forms a ternary complex with odin and EphA8, and RINL affects the degradation of the EphA8 receptor.

<p>(A) HEK293T cells were co-transfected with EphA8-FLAG, HaloTag-odin, and myc-RINL (+) or mock (−) plasmids as indicated, and cell lysates were immunoprecipitated with anti-myc antibody. Immunoprecipitated fractions and total lysates were immunoblotted with antibodies as indicated. (B and C) HeLa cells were transfected with EphA8-FLAG and myc-RINL or mock plasmids, and total lysates were immunoblotted with antibodies as indicated. ΔSH2; SH2 domain-deleted mutant. The data obtained from three independent experiments are shown (C) as the mean ± S.E. (error bars). *, p<0.05 vs. mock-transfected cells. N.S., not significant. (D and E) HEK293T cells were transfected with EphA8-FLAG and myc-RINL or mock plasmids, and total lysates were immunoblotted with antibodies as indicated. WT; wild type. The data obtained from three independent experiments are shown (E) as the mean ± S.E. (error bars). *, p<0.05 vs. mock-transfected cells. (F and G) HeLa cells were transfected with 30 pmol scrambled negative control (NC) or RINL-specific siRNA. 24 hours after the transfection, these cells were transfected with EphA8-FLAG and siRNA-resistant FLAG-RINL, and incubated for 48 hours. Total proteins from the cell lysates were subjected to SDS-PAGE and immunoblotted (IB) with antibodies as indicated. The data obtained from three independent experiments are shown (G) as the mean ± S.E. (error bars). **, p<0.01 vs. NC-transfected cells. *, p<0.05 vs. siRNA-transfected cells with FLAG-mock plasmid transfection. (H and I) HeLa cells were transfected with EphA8-FLAG and FLAG-RINL (+, lanes 2–5) or mock plasmids (−, lane 1), and total lysates were immunoblotted with antibodies as indicated. These cells were non-treated (NT, lanes 1 and 2), or treated with MG132 (20 µM, lane 3), leupeptin (100 µg/ml, lane 4), or bafilomycin (200 nM, lane 5) for 3 hours. Total lysates were immunoblotted with antibodies as indicated. The data obtained from three independent experiments are shown (I) as the mean ± S.E. (error bars). **, p<0.01 vs. mock-transfected cells. *, p<0.05 vs. non-treatment cells transfected with RINL.</p