Wild type and point mutants that lost GEF activities for Rab5 were transiently co-transfected with myc-odin into HEK293T cells. RIN2 and RIN3 exhibit GEF activities for Rab22 (Physique S1). Similarly, Rabex-5 has been reported to exhibit 100-fold lower GEF activity for Rab22 than for Rab5 and Rab21 and BL21-CodonPlus (DE3)-RIL (Stratagene) by glutathione Sepharose 4B resin (GE Healthcare). In vitro guanine nucleotide-binding assay The GTPgammaS-binding assay was performed using the filter method as described previously [6]. Briefly, GST-Rab5, Rab21, Rab22, and Rab31 (2.0C3.5 pmol GTPgammaS-binding activity) were incubated with 1 M [35S]GTPgammaS (20,000 cpm/pmol) at 30C for the indicated times in the presence or absence of FLAG-RINL, RIN3, or Rabex-5 purified from baculovirus-infected Sf9 cells. Radiolabeling of nucleotides associated with the Rab family in intact cells and identification of nucleotide-bound forms HEK293T were transfected with myc-Rab5 subfamily proteins and FLAG-Rab5-GEF proteins. Guanine nucleotides associated with myc-Rab5 subfamily were analyzed as described previously [12], [24]. Yeast two-hybrid screening A yeast two-hybrid assay was performed as described previously [6]. The yeast reporter-strain Hf7c was transformed with pGBT9-RINL using a lithium acetate-based method and produced in synthetic medium lacking tryptophan at 30C for 3 days. The cells were transformed with the mouse brain MATCHMAKER cDNA library (Clontech) and plated on synthetic medium lacking leucine, tryptophan, and histidine at 30C for 5 days. For conversation analyses, transformed yeasts were lifted onto filter papers, lysed in liquid nitrogen, and incubated with 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside. Supporting Information Physique S1 GEF activity of the RINL for the Rab5 subfamily. (ACF) The purified GST-Rab5a (A, 3.5 pmol of alive GTPgammaS-binding activity), Rab5b (B, 2.5 pmol), Rab5c (C, 3 pmol), Rab21 (D, 2 pmol), Rab22 (E, 3 pmol), or Rab31 (F, 2 pmol) was incubated at 30C with 1 M [35S]GTPgammaS for the indicated occasions in the presence of 8 pmol of FLAG-RINL (filled squares), Rabex-5 (filled triangles) or FLAG peptide alone (open circles). The amounts of [35S]GTPgammaS bound to the Rab5 subfamily are illustrated as the functions of the incubation occasions. (TIF) Click here for additional data file.(2.8M, tif) Physique S2RIN2 and RIN3 exhibit GEF activities for Rab22 em in vitro /em . GST-Rab22 (2 pmol of alive GTPgammaS-binding activity) was incubated at 30C with 1 M [35S]GTPgammaS for the indicated occasions in the absence (Rab alone) and presence of 8 pmol of RIN1 (filled squares), RIN2 (filled diamonds), RIN3 (filled circles) or FLAG-Rabex-5 (filled triangles). No [35S]GTPgammaS-binding activity was detected in the fractions of the RIN family or Rabex-5 (data not shown). (TIF) Click here for additional data file.(576K, tif) Physique S3Diagram of the structural features of the odin/Anks1a. The numbers represent the amino acid residues. cDNA coding 583C1150 amino acids of odin was identified to interact with RINL in Rabbit polyclonal to ETFA beta-galactosidase assay by yeast two-hybrid system. (TIF) Click here for additional data file.(794K, tif) Physique S4RINL interacts with odin independent of its GEF activity. Wild type and point mutants that lost GEF activities for Rab5 were transiently co-transfected with myc-odin into HEK293T cells. Cells lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-myc and anti-FLAG antibodies. Aliquots of total lysates were also immunoblotting Betaxolol hydrochloride with anti-myc antibody. (TIF) Click here for additional data file.(507K, tif) Physique Betaxolol hydrochloride S5Diagrams of deletion mutants of RINL and odin. The numbers represent the amino acid residues. (TIF) Click here for additional data file.(821K, tif) Physique S6EphA8 stably expressing in Neuro2A cells is degraded by the expression of RINL. Neuro2A cells stably expressing EphA8-HA are transfected with myc-mock, RINL/WT, or RINL/YT_AA for 24 hours, and total lysates from these cells were immunoblotted with antibodies as indicated. (TIF) Click here for additional data file.(506K, tif) Acknowledgments We thank Hideko Uga for generating anti-odin monoclonal antibody. We also would like to thank all experts who participated in this study: Shinya Takahashi, Manabu Yoshikawa, Kyoko Sakurai, Arisa Ebihara, Seiichi Ikeda, and Satoshi Nakagawa. Footnotes Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported in part by research grants (to H.K., K.K. and T.K.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and Japan Society for the Betaxolol hydrochloride Promotion of Science (JSPS) (URL: http://www.mext.go.jp/english/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study..
