HUMAN RASL HUMAN RLNL MOUSE RASL MOUSE RMNL RAT RASL RAT RMHL CONSENSUS RXXL AC B Supplement Fig. 1 Supplement Figure 1 Sequence features of FLJ25439 protein. (A) Conservation of D-box in human and rodent FLJ Among three D-boxes identified in human FLJ25439, two ( aa and aa) are conserved in mouse and rat while the third is not found in rodents. Signature motif of D-box is indicated in red. (B) Amino acid sequence of tetratricopeptide repeat (TPR) domain in human FLJ Red denotes amino acid homologous to consensus sequence. (C) Phylogeny of FLJ25439 shows closer relationship of human FLJ25430 to canine (C.familiaris) than to rodents (M. musculus and R. norvegicus).

+peptide - peptide Tubulin 2A4 DAPI Merge ABCD EFGH Supplement Fig. 2 Supplement Figure 2 Immunofluorescent micrographs demonstrate the specificity of 2A4 monoclonal antibody against FLJ25439 protein. HeLa cells were fixed and immunostained with antibodies to FLJ25439 (2A4, green), ß-tubulin (red), and nuclei counter stained with DAPI in the presence (+) or absence (-) of immunogenic peptide for antibody generation. Note the localization of FLJ25439 at midbody (insets in F,H) but not detectable in the same structure in the presence of peptide (inset in D). Bar, 5μm

Supplement Fig. 3 ABCDEF GHIJ K L M N OPQR S T UV W X Supplement Figure 3 Immunofluorescent micrographs of endogenous FLJ25439 protein localization during cell cycle progression in HeLa cells. HeLa cells were fixed and immunostained with antibodies to FLJ25439 (red), ß-tubulin (green), r-tubulin (green) Cep55 (green) and nuclei counter stained with DAPI. FLJ25439 is observed specifically at midbody where it colocalizes with Cep55 (F, L, X). r-tubulin denotes the location of centrosomes (G) where little or no staining of FLJ25439 can be observed (A and S). The insets represent enlarged region pointed by arrows. Bar, 5μm

Supplement Fig. 4 ABCDEF G H I J K L MN O P Q R STU V W X Supplement Figure 4 Immunofluorescent micrographs of exogenously expressed FLJ25439 protein localization during cell cycle progression in HeLa cells. HeLa cells were transfected with HA-tagged FLJ25439, fixed and immunostained with antibodies to HA(red), ß-tubulin (green) r-tubulin (grees), Cep55 (green) and nuclei counter stained with DAPI. Exogenously expressed FLJ25439, identified by anti-HA labeling, is observed at centrosomes(G and S) as well as at midbody (arrows in F and X) in cytokinesis. r-tubulin denotes the location of centrosome (G) while Cep55 denotes midbody position (L). The insets represent enlarged region pointed by arrows. Bar, 5μm

Supplement Fig. 5 Supplement Figure 5 PSD analysis of GRP78 peptide fragment. A MALDI-TOF spectrum of GRP78 and the parent ions m/z , and were selected for further analysis by an UltraflexTM MS/MS operated in the LIFT mode using FlexControl TM software. A sequence was confirmed from the labeled b- and y-ions in the spectrum.

Supplement Fig. 6  -actin Grp78 HeLa HeLa (1-16) HeLa-pOZ Supplement Figure 6 GRP78 expression levels of parental cell (HeLa), mock cell (HeLa-pOZ) and FLJ25439 stable clone (HeLa 1-16). The plasmid pOZ-transfected cells as a mock control did not result in promotion of GRP78 level while GRP78 was upregulated in FLJ25439-transfected HeLa cells, suggesting that the vector would not markedly affect cell physiology in our cell model.